RU2746410C2 - Mutant microorganisms resistant to death under the action of lactose - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to a method for producing microorganisms resistant to the phenomenon of suppression by the action of lactose, to microorganisms obtained by using this method. A method for producing microorganisms resistant to the phenomenon of lactose suppression, grown in an environment in which lactose is combined with another carbon source, involves changing the expression of the lactose transporter in microorganisms by introducing a heterologous promoter in front of an endogenous or exogenous lactose transporter gene or by changing the codons of the endogenous lactose transporter gene to codons used by the microorganism more often, or to codons used by the microorganism less often, the subsequent cultivation of these microorganisms on a medium that includes a carbon source other than lactose, as well as the addition of lactose to the specified medium, including a carbon source other than lactose, during the growth of these microorganisms and the detection of the lactose transporter expression. Selection of microorganisms resistant to the phenomenon of suppression by the action of lactose, growing on the specified medium, including lactose and another carbon source, which retain at least 50% of the level of lactose transport into the cell, achieved with the wild-type expression cassette of the specified lactose transporter, is carried out on based on the detected expression of the lactose transporter. At the same time the specified carbon source other than lactose is selected from the group consisting of glycerin, maltose, glucose, fructose, sucrose, fucose, mannose, sialic acid, starch, cellulose, polyols, organic acids and pentoses. Also proposed are variants of microorganisms obtained by this method. An expression cassette of a lactose transporter is also proposed, leading to the expression of a lactose transporter, which does not lead to the phenotype of suppression by the action of lactose, which is isolated from a variant of the specified microorganism. he specified expression cassette and the specified variants of microorganisms are used for the production of 2-fucosyllactose, 2'-fucosyllactose, 3-fucosyllactose, 2', 3-difucosyllactose, lacto-N-triose, lacto-N-tetraose, lacto-nN-tetraose, 3'-sialyllactose, 6'-sialyllactose, glycolipids or galactosylated compounds. A method for producing an expression cassette is also proposed.

EFFECT: invention provides for the preservation in the resulting microorganism of at least 50% of the level of lactose transport into the cell, achieved with the wild-type expression cassette of the specified lactose transporter, based on the detected expression of the lactose transporter.

14 cl, 1 tbl, 21ex, 21 dwg

Description

Technology area

The present invention relates to a method for producing mutated microorganisms resistant to the phenomenon of death under the influence of lactose, and to microorganisms obtained by this method. Such engineered microorganisms can be used for the production of specialty products, including, for example, special carbohydrates, glycolipids and galactosylated compounds, but not limited to them.

State of the art

Death by lactose is a well-known and well-studied pattern, according to which the growth of many organisms is suppressed in the presence of lactose and another carbon source. The exact mechanism underlying this phenomenon, however, remains unknown, although it is clear what is required to trigger this mechanism. Death by lactose occurs when lactose is added to a microbial culture growing on a different carbon source, such as, but not limited to, glycerol or sucrose. In addition, this occurs when the lactose transporter gene is either induced or constitutively expressed (33, 39, 75). For the first time, death under the influence of lactose was observed in E. coli, when the expression of lactose permease was modulated by IPTG and lactose under chemostat conditions (28). Later, death under the action of lactose was also observed in Rhizobium meliloti, Kluyveromyces lactis, and Zymomonas mobilis (55, 70, 77). One of the possible reasons for this phenomenon was called the "high price" of the activity of the lactose transporter for the cell; to compensate for such high costs, a decrease or inhibition of growth occurred, in addition, this is also associated with the extracellular concentration of lactose (34), which is maintained high in most technological processes. to obtain sufficiently high titers and product yields. However, it is believed in the art that as long as lactose is transported under these conditions, death by lactose will also occur. To solve the problem of death under the influence of lactose, it was proposed to delete lactose permease or severely disrupt the absorption of lactose (34). For example, Lodi et al. (55) deleted (knocked out) the lactose permease gene in K. lactis and found that death under the action of lactose was no longer observed. In the course of these experiments, it was also shown that spontaneous mutations led to the emergence of strains negative for the phenomenon of death by lactose, which were found to have strong disturbances in the absorption of lactose. However, the absorption of lactose is essential for the efficient synthesis of specialty foods or bioproducts. Thus, it is obvious that deletion of lactose permease or severe disruption of lactose permease activity is not a solution, since the production of such bioproducts requires 1) efficient absorption of lactose and 2) the presence of an expression cassette, which is not associated with the "death phenotype by lactose". Lactose permease has already been used in the past for the production of lactose-based bioproducts, but without solving the problem of the “death phenotype by lactose”. In the past, this problem has been addressed either by dramatically reducing the absorption of lactose (and thus resulting in few or no lactose-based specialty foods being produced) or by decoupling the growth phase from the production phase in order to build up sufficient amount of biomass. After the end of this growth phase, lactose was added to the second phase to obtain a special product. In this second phase, growth stops or decreases markedly (59).

Thus, at present, in order to avoid death by lactose, the absorption of lactose must be stopped or severely impaired, since the normal absorption of lactose will always lead to death by lactose. On the contrary, the present invention discloses a screening technique for the detection of lactose permease expression cassettes that would provide efficient uptake of lactose without subsequent death by lactose.

Lactose is a building block for many bioproducts, more specifically for specialty carbohydrates (14), glycolipids, and galactosylated compounds such as galactosillated lipids, galactosylceramides and galactosylated aglycones. Galactose residues are often used for targeted delivery of pharmaceutical agents to specific organs (28). The use of lactose as a substrate in combination with other substances, however, is not as obvious as it might seem, due to the phenomenon of "death by lactose" described above. In most cases, to avoid the phenomenon of death under the influence of lactose, it is necessary to use multiphase systems of obtaining, conjugated cellular systems of non-growing cells (32, 48).

The basic structural framework of many specialty carbohydrates consists of lactose or galactose residues. More specifically, human milk oligosaccharides, and therefore breast milk oligosaccharides, an extensive group of saccharides and oligosaccharides, are built from galactose and lactose residues (15). These carbohydrates are further modified with sugar residues such as, for example, N-acetylglucosamine, N-acetylgalactosamine, sialic acids (such as N-acetylneuraminate, N-glucoyl neuraminate, 2-keto-3-deoxy-D-glycero-galacto-nonulosonic acid and others) (21), L-fucose and others. The synthesis of these compounds requires activated carbohydrates, such as UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-sialic acid, GDP-fucose and others, which are expensive and difficult to synthesize, but which are produced optimally by living growing cells. since energy is required for their biosynthesis.

Oligosaccharide components of human / breast milk have anti-inflammatory and prebiotic effects and / or can be used for therapeutic purposes as a nutraceutical, anti-inflammatory agent, prebiotic, or pharmaceutical agent (15, 24, 68). Nevertheless, there is still a need to develop an effective method for obtaining the listed high-value compounds.

The present invention describes synthetic expression systems for lactose transporters that do not result in death by lactose even at high lactose concentrations. Mutated organisms containing these expression systems can thus be used to produce the bioproducts described above.

Brief Description of Drawings

Figure 1. Effect of lactose on wild type E. coli. The arrow indicates the moment of adding lactose to one of the cultures, as a result of which the growth of this culture immediately stopped, while the other strain continued to grow in another culture.

Figure 2. The effect of lactose on the E. coli strain, mutant in the lactose transporter gene, in which the expression of the lactose transporter is altered by a synthetic constitutive promoter. The arrow indicates the moment of adding lactose to the medium in one of the cultures. In this case, it had no effect on growth. Thus, these mutant strains can be selected in this way.

Figure 3. An example of a combination of promoter, RBS and lactose transporter sequence (SEQ ID No. 1), for which, when introduced into a mutant strain of E. coli, no death is observed under the action of lactose.

Figure 4. Example of the sequence of the lactose permease gene, translationally linked to the lacZ gene (SEQ ID No. 2).

Figure 5. Example of the sequence of the lactose permease gene lacY, translationally linked to the cat gene (SEQ ID NO. 3).

Figure 6. Example of the sequence of the K. maxianus lactose permease gene, translationally linked to the aph 1 gene (SEQ ID No. 4).

Figure 7. Example of the sequence of the lactose permease lacy gene associated with an aptamer that binds to (Z) -4- (3,5-difluoro-4-hydroxybenzylidene) -1,2-dimethyl-1H-imidazole-5 (4H) - onom (SEQ ID No. 5), making it possible to detect the expression of lactose permease.

Figure 8. Chloramphenicol resistance of the reference strain containing the reference plasmid pSC101 without the lactose permease gene, translationally linked to the chloramphenicol resistance gene, and the mutant strain with the lactose permease gene, translationally linked with the chloramphenicol resistance gene. The X-axis plots the various concentrations of chloramphenicol tested, the Y-axis plots the absorbance values of the culture after 48 hours of incubation. The comparison strain demonstrates growth retardation at lower chloramphenicol concentrations during screening compared to the mutant strain, which confirms that screening for the expression of lactose permease can be carried out by translationally linking its gene to the antibiotic resistance gene. Therefore, organisms expressing lactose permease can be selected from a mixture of non-expressing and expressing organisms in the manner described.

Figure 9. Chloramphenicol resistance of the reference strain containing the reference plasmid pSC101 without the lactose permease gene, translationally linked to the chloramphenicol resistance gene, and the mutant strain with the lactose permease gene, translationally linked to the chloramphenicol resistance gene. The X-axis plots the various tested concentrations of chloramphenicol, the Y-axis plots the absorbance values of the culture after 92 hours of incubation. The comparison strain demonstrates growth retardation at lower chloramphenicol concentrations during screening compared to the mutant strain, which confirms that screening for the expression of lactose permease can be carried out by translationally linking its gene to the antibiotic resistance gene. Therefore, organisms expressing lactose permease can be selected from a mixture of non-expressing and expressing organisms in the manner described.

Figure 10. pCXP14-FT_H. pylori (SEQ ID No. 6).

Figure 11. Effect of lactose on wild-type yeast strain (Kluyveromyces marxianus lactis). The arrow indicates the moment of adding lactose to one of the cultures, as a result of which the growth of this culture immediately stopped, while the other strain continued to grow in another culture.

Figure 12. The effect of lactose on yeast strains (Saccharomyces cerevisiae) with a mutant lactose transporter, in which the expression of the lactose transporter is altered by a synthetic constitutive promoter. The arrow indicates the moment of adding lactose to the medium in one of the cultures. In this case, it had no effect on growth. Thus, these mutant strains can be selected in this way.

Figure 13. Example of a combination of a promoter (p1), a Kozak sequence, a K. marxianus lactose permease coding sequence and a terminator (SEQ ID NO. 7), for which no lactose death is observed when introduced into a mutant yeast strain.

Figure 14. Example of a combination of a promoter (p2), a Kozak sequence, a K. marxianus lactose permease coding sequence and a terminator (SEQ ID NO. 8), for which no lactose death is observed when introduced into a mutant yeast strain.

Figure 15. Example of a combination of a promoter, a Kozak sequence, a K. marxianus β-galactosidase coding sequence and a terminator (SEQ ID No. 9).

Figure 16. HR1 rDNA (SEQ ID NO. 10).

Figure 17. HR2 rDNA (SEQ ID NO. 11).

Figure 18. Examples of selected sequences found using the screening method for the absence of death by lactose as described in the examples (SEQ ID No. 12-57).

Figure 19. Relative growth rate of lactose permease expression cassettes, compared to wild type. The whiskers of the graph represent the standard deviation of at least 3 repeated measurements. The sequences corresponding to the X-axis sequence numbers are shown in Figure 18.

Figure 20. Sequence of the lacIQ_lacY expression cassette that was tested for death by lactose in Example 16 (SEQ ID No. 58).

Figure 21. Effect of lactose on wild-type E. coli and placIQ_lacY mutant strain. Both strains were grown with and without added lactose in the middle of the exponential phase. The arrow indicates the time of addition of lactose. The growth of both strains was severely impaired by the addition of lactose.

The essence of the invention

The present invention relates to a method for producing lactose-resistant microorganisms grown in an environment in which lactose is combined with another carbon source, said method comprising:

a. mutating the expression of lactose transporters in microorganisms, where said mutation results in an expressed lactose transporter,

b. growing said mutated microorganisms on a medium containing a carbon source other than lactose,

c. adding lactose to said medium during the growth of said mutated microorganisms, and

d. selection of microorganisms resistant to the phenomenon of death under the influence of lactose, growing on the specified medium, including lactose.

More specifically, the present invention relates to a method for producing lactose-resistant microorganisms grown in an environment in which lactose is combined with another carbon source, said method comprising:

a. mutating the expression of lactose transporters in microorganisms, where said mutation results in the expression of said lactose transporter

b. growing said mutated microorganisms on a medium containing a carbon source other than lactose,

c. adding lactose to said medium during the growth of said mutated microorganisms, and

d. selection of microorganisms resistant to the phenomenon of death under the influence of lactose, growing on the specified medium, including lactose, and which retain at least 50% of the level of transport of lactose into the cell, achieved with the expression cassette of the wild type of the specified lactose transporter.

The present invention further relates to a method as defined above, wherein step a) is carried out by introducing a heterologous promoter upstream of an endogenous or exogenous lactose transporter gene, and / or by mutating an untranslated region in front of a coding sequence that contains a ribosome binding sequence or a Kozak sequence, and / or by modifying the frequency of codon use in the endogenous lactose transporter gene.

The present invention also relates to a method as described above, wherein said introduction of a heterologous promoter upstream of an endogenous or exogenous lactose transporter gene is accomplished by a) deleting endogenous lactose transporters from the genome and introducing them into another region in the genome of said microorganism, or b) by introducing a heterologous promoter in front of the endogenous lactose transporters, or c) by knocking out the endogenous lactose promoter and introducing the heterologous promoter into the same region of the genome of said microorganism.

The present invention further relates to the method described above, wherein the expression of the lactose permease in step b) is detected by translational binding to a reporter gene and / or by binding to an aptamer.

The present invention relates to the method described above, wherein the expression of the lactose transporter is detected by the genetic constructs shown in SEQ ID NOs 2, 3, 4 and / or 5.

The present invention further relates to the method described above, wherein said lactose transporter is lactose permease.

The present invention further relates to the method described above, wherein said microorganism is a bacterium, yeast or fungus.

The present invention also relates to promoter sequences, untranslated regions upstream of the coding sequence that contain a ribosome binding sequence or a Kozak sequence, and / or lactose permease sequences realizing the expression of a lactose transporter that does not lead to the phenotype of death by lactose when a microorganism containing such sequence, grows in an environment in which lactose is combined with other carbon source (s), and obtained in the manner described above.

The present invention also relates to a microorganism resistant to death by lactose, grown in an environment in which lactose is combined with another carbon source, and obtained by the method described above.

More specifically, the present invention relates to a microorganism as described above and containing a heterologous sequence upstream of the lactose transporter gene shown in SEQ ID NOs 1, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46.47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57.

The present invention also relates to a microorganism resistant to the phenomenon of death by lactose, in which the genes encoding enzymes from the pathways of lactose and / or galactose degradation are less functional or non-functional.

The present invention also relates to the use of the microorganism described above for the manufacture of specialty lactose-based products such as specialty carbohydrates, glycolipids and galactosylated compounds.

The present invention further relates to the use of a microorganism as described above, wherein said special carbohydrates are 2-fucosyllactose, or 2'-fucosyllactose, or 3-fucosyllactose, or 2 ', 3-difucosyllactose, or lacto-N-triose, or lacto- N-tetraose, or lacto-N-tetraose, or 3'-sialyllactose, or 6'-sialyllactose.

Detailed description of the invention

The present invention describes a new method for avoiding death by lactose by altering the expression of the lactose transporter through the use of a genetically engineered organism, a mutant organism expressing a lactose transporter. For this purpose, the exogenous and / or endogenous gene of the lactose transporter is expressed using a heterologous promoter, which does not lead to the death phenotype under the action of lactose, and / or the frequency of using the codons of the lactose transporter is modified in order to obtain an altered expression of the lactose transporter, which does not lead to the death phenotype. under the influence of lactose. For this, the natural system for controlling the expression of lactose transporters must be removed and / or replaced in such a way that the phenomenon of death under the influence of lactose is not observed. For example, a naturally occurring lactose transporter expression cassette is removed from the genome and reintroduced at another site, and / or a heterologous promoter is introduced upstream of the lactose transporter at its original location, and / or the lactose transporter is first knocked out and then introduced with the heterologous promoter into the same region of the genome and / or in another region of the genome, and / or the lactose transporter is introduced into the operon, which is expressed under the action of the heterologous promoter.

The present invention thus relates to a method for producing lactose-resistant microorganisms grown in an environment in which lactose is combined with another carbon source, said method comprising: 1) mutating the expression of lactose transporters in microorganisms, wherein said mutation leads to the expression of the specified lactose transporter, 2) the cultivation of these mutated microorganisms on a medium including a carbon source other than lactose, 3) the addition of lactose to the specified medium during the growth of these mutated microorganisms and 4) selection of microorganisms resistant to the phenomenon of death under the influence of lactose growing on the specified medium, including lactose, and which retain at least 50% of the level of transport of lactose into the cell, achieved with the expression cassette of the wild type of the specified lactose transporter.

The term "death by lactose" refers to the phenomenon of stunting or stopping growth of an organism that is grown in an environment in which lactose or galactoside is combined with other carbon source (s). Carbon sources are glycerin, maltose, glucose, fructose, sucrose, fucose, mannose, sialic acid, starch, cellulose, polyols (such as mannitol, xylitol, sorbitol), organic acids (lactate, succinate, acetate, and others) and / or pentose (xylose, arabinose and others), but not limited to this.

The present invention describes a method for identifying lactose permease expression systems that are not killed by lactose. This method involves analyzing the growth of a mutant strain to which lactose is added in the middle of the exponential phase. This method can be carried out in a high-throughput format in microtiter plates or using cell sorters that screen for multiple promoters, ribosome binding sites, codon frequency, and other factors that may affect the expression of lactose permease in a mutant microorganism. The present invention describes a method for detecting the expression of lactose permease by translationally binding and / or binding to an aptamer and selecting sequences that lead to expression using a reporter gene, such as, for example, an antibiotic resistance gene (for example, chloramphenicol, geneticin G418, but not limited to), a fluorescent protein gene, a hydrolase gene (eg, galactosidase, xylanase, but not limited to) and / or a sequence of aptamer, but not limited to them. Sequences leading to the expression of lactose permease are then selected using a reporter gene by growing on antibiotic media, colorimetrically (e.g., X-gal), and / or using a fluorescence activated cell sorter that sorts fluorescent cells, and / or using aptamer analysis, for example, using (Z) -4- (3,5-difluoro-4-hydroxybenzylidene) -1,2-dimethyl-1H-imidazol-5 (4H) -one (37, 64), but not limited to this. The present invention further describes a screening technique for detecting mutant organisms expressing a lactose transporter that are not killed by lactose. In addition, the present invention describes the creation of lactose permease expression cassette libraries from promoter libraries, RBS or Kozak sequence libraries, transcription terminator libraries and / or lactose permease gene variants with different codon frequencies. These libraries can also be created using such methods as, but not limited to, Gibson assembly, Golden Gate assembly, Cliva assembly, LCR, or restriction and ligation (25, 36, 50, 79).

The term "which retain at least 50% of the level of lactose transport into the cell achieved with the wild-type expression cassette of said lactose transporter" refers to the fact that the mutated microorganisms of the present invention, although they are resistant to death under action of lactose should retain at least 50% (i.e. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%) of the level of lactose transport into the cell achieved with expression cassette wild type of the specified lactose transporter.

The term "expression cassette" refers to any sequence in which a promoter sequence, a untranslated region sequence (containing a ribosome binding sequence or Kozak sequence), a coding sequence (eg, a lactose permease gene sequence) and optionally a transcriptional terminator are present (18).

The term "lactose transporter" refers to any protein expressed in a microorganism that is capable of translocating (transporting) lactose across the cytoplasmic membrane. Such proteins are, for example, lactose permeases (transporters from the MFS superfamily or the Major Facilitator Superfamily of membrane transporters).

The term "heterologous promoter" refers to any promoter that does not occur naturally upstream of the coding sequence. "Promoter" is the complete RNA polymerase binding sequence upstream of the transcription start site and the untranslated region upstream of the coding sequence. A "heterologous promoter" sequence is thus 1) a variant of a naturally occurring promoter sequence containing at least 1 mutation (i.e. 1, 2, 3, 4 or more), and / or 2) native a promoter from a mutant microorganism expressing a lactose transporter that does not occur naturally before the coding sequence of said transporter, and / or 3) a sequence that does not occur naturally in a microorganism expressing a lactose transporter, and / or 4) an artificial promoter that is in silico constructed promoter. These promoters can be found in libraries such as those described by Alper et al. (2005), and / or HXT7p, Hammer et al. (2006), De Mey et al. (2007), Coussement et al (2014) or Mutalik et al (2013) (3, 25, 29, 41, 60), (66), or Blount et al (2012), may be promoters such as ADH1p, TEF1p, TEF2p, GPDp, PDC1p, FBA1p, PGK1p, PGI1p, TDH2p, PYK1p, ENO2p, GPM1p, TPI1p (13), or promoters described, for example, by Rhodius et al. (2012). The term "artificial promoter" also refers to promoters with DNA sequences that are combinations of the sequence of the native (autologous) promoter with portions of the sequences of other (autologous or heterologous) promoters. The sequences of such “artificial promoters” can be found in databases such as, for example, partsregistry.org (19). Heterologous promoters result in either constitutive expression or regulated expression via a transcription factor.

The term "constitutive expression" means expression that is not regulated by transcription factors other than RNA polymerase subunits (eg, bacterial sigma factors) under certain growth conditions. Non-limiting examples of such transcription factors are CRP, LacI, ArcA, Cra, IclR and others in E. coli or Aft2p, Crz1p, Skn7 and others in Saccharomyces cerevisiae, or DeoR, GntR, Fur and others in B. subtilis. These transcription factors bind to a specific sequence and can block or enhance expression under specific growth conditions. RNA polymerase binds to a specific sequence to initiate transcription, for example, via sigma factor in prokaryotic cells.

The term "regulated expression" means expression that is regulated by transcription factors other than RNA polymerase subunits (eg, bacterial sigma factors) under certain growth conditions. Examples of such transcription factors are described above.

The term "untranslated region" before a coding sequence that contains binding sites for the ribosome or Kozak sequence refers to the sequence between the binding sequence of the RNA polymerase and the coding sequence. Such untranslated region is also a sequence that naturally occurs before the coding sequence and / or a sequence that occurs naturally in a microorganism expressing a lactose transporter and / or a sequence obtained from other organisms (prokaryotes or eukaryotes) and / or artificially an engineered sequence that refers to unnatural or in silico engineered ribosome binding sites at a known or measurable translation rate, these sequences can be obtained from libraries as described by Mutalik et al. (2013) (60), or created using algorithms, for example, as described by Salis et al. (2009) (67), or can be found in databases such as partsregistry.org (19).

The term "modified codon frequency" refers to changing the codons used in the DNA coding sequence, either to codons used by the body more often, or to codons used by the body less frequently. Codon frequency is determined using databases such as a codon frequency database (61), and codon frequency is optimized using special codon frequency optimization algorithms (73).

The term "translationally linked" refers to the binding of the expression of a target gene to the expression of a reporter gene, for example, a green fluorescent protein gene, an antibiotic resistance gene, a toxin gene (49, 53, 58, 63). The term "translational sensor" refers to any mechanism that can be indicative of gene expression and translation, for example, fluorescent labels and cleaved labels described in the following references (17, 72).

The term "binding to aptamer" refers to the introduction of the sequence of the aptamer into the messenger RNA of the lactose transporter, which can be detected with a fluorophore such as (Z) -4- (3,5-difluoro-4-hydroxybenzylidene) -1, 2-dimethyl-1H-imidazol-5 (4H) -one (37, 64).

The term "growth analysis" refers to the analysis of the growth curve of an organism. This organism is cultivated in a growth medium under growth conditions. Growth condition refers to all environmental parameters such as pH, temperature, dissolved oxygen. The pH is adjusted using a pH buffer or by controlling the pH and is, for example, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8, but not limited to this. The temperature is set to, for example, but not limited to 25, 28, 30, 32, 34, 37, 40, 42, 45 ° C. The dissolved oxygen level corresponds to anaerobic, micro-aerobic (dissolved oxygen concentration less than 1.5 mg / l), or aerobic conditions.

The term "medium" or "growth medium" refers to any solution containing substances necessary for the body to grow. These substances are nitrogen sources such as ammonium salts, nitrates, yeast extract, peptone, casamino acids and / or amino acids, but not limited to, phosphorus sources such as, but not limited to, phosphates, sulfur sources such as sulfates, but but not limited to, elements such as, but not limited to, copper, cobalt, iron, selenium, iodine, molybdate, magnesium, calcium, potassium, sodium, zinc, nickel, manganese, and / or boric acid, and / or vitamins, such as but not limited to thiamine, pantothenic acid and / or niacin and / or carbon sources such as glycerin, maltose, glucose, fructose, sucrose, fucose, mannose, sialic acid, starch, cellulose, polyols (such as mannitol, xylitol, sorbitol), organic acids (lactate, succinate, acetate and others), and / or pentose (xylose, arabinose and others), but not limited to them.

The term "organism" or "cell", as indicated above, refers to a microorganism selected from the list of bacteria, yeast or fungi, or refers to a plant or animal cell. The bacteria preferably belong to the phylum Proteobacteria, or the phylum Firmicutes, or the phylum Cyanobactria, or the phylum Deinococcus-Thermus. The bacteria belonging to the phylum Proteobacteria preferably belong to the Enterobacteriaceae family, preferably the species Escherichia coli. The bacteria preferably refers to any strain belonging to the species Escherichia coli, for example, including but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term refers to cultured Escherichia coli strains, designated E. coli K12 strains, which are well adapted to the laboratory environment and, unlike wild-type strains, have lost their ability to grow in the intestine. Well-known examples of E. coli K12 strains are wild type K12, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Thus, the present invention particularly relates to a mutated and / or transformed Escherichia coli strain as defined above, wherein said E. coli strain is a K12 strain. More specifically, the present invention relates to a mutated and / or transformed Escherichia coli strain as defined above, wherein said K12 strain is E. coli MG1655. The bacteria belonging to the phylum Firmicutes preferably belong to the Bacilli, preferably the Bacillus species. The yeast preferably belongs to the phylum Ascomycota, or the phylum Basidiomycota, or the phylum Deuteromycota, or the phylum Zygomycetes. The yeast preferably belongs to the genus Saccharomyces, Pichia, Hansunella, Kluyveromyces, Yarrowia or Starmerella. The fungi preferably belong to the genus Rhizopus, Dictyostelium or Aspergillus.

The present invention describes organisms that are capable of absorbing lactose and not being killed by lactose and that are capable of converting lactose or galactose residues in its composition into a special product. More specifically, said specialty products or bioproducts are specialty carbohydrates, glycolipids, including but not limited to galactolipids and / or lactolipids, and / or galactosylated compounds such as galactosillated lipids, galactosylceramides and / or galactosylated aglycones.

The present invention describes organisms that are capable of absorbing lactose without being killed by lactose, and converting said lactose into human milk oligosaccharides such as 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2 ', 3-difucosyllactose, 2 ', 2-difucosyllactose, 3,4-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6-disialyllactose, 6,6'-disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose -tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a , lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosyl monosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose , monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto-N-hexaose aose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated / oligosaccharized and oligosaccharized but not limited to them.

The present invention further describes organisms that are not killed by lactose and that synthesize sugars that make up the nucleotides, such as GDP-L-fucose, GDP-mannose, GDP-glucose, CMP-sialic acid, UDP-glucose, UDP- galactose, UDP-N-acetylglucosamine, UDP-N-acetylmannosamine, UDP-N-acetylgalactosamine, UDP-glucuronic acid, UDP-galacturonic acid, UDP-xylose, UDP-arabinose and / or dTDP-rhamnose, but not limited to them. The term "sialic acid", as defined by Varki (1992) (71), refers to a group of compounds such as, but not limited to, neuraminic acid, N-acetylneuraminic acid or N-glycosylneuraminic acid. The intracellular concentration or pool of GDP-fucose increases due to upregulation of the metabolic pathway of de novo synthesis and / or increased reutilization. The de novo metabolic pathway consists of GDP-4-keto-6-deoxymanose-3,5-epimerase-4-reductase or GDP-fucose synthase, GDP-mannose 4,6-dehydratase, GDP-D-mannose pyrophosphorylase, phosphomannose isomerase and / or phosphomannomutases, the expression of which is individually increased by genetic elements such as, but not limited to, promoters and / or ribosome binding sites, and / or by changing the frequency of codon use in the monocistron or polycistron (operon structure), and / or with the regulators arcA iclR in Enter obacteriaceae, and / or with the transcriptional regulator RcsA in Enterobacteriaceae or homologous genes in bacteria, or Xbp1, Spt20, Sfp1, Rpd3, Rap1, Gcr1, Gcn5, Cst6, Abf1, Hsf1, Reb Sin4, Ash1, Ixr1, Met32, Pho2, Rgr1, Spt7, Swi4 in Saccharomyces cerevisiae, or homologous genes in yeast or fungi. The metabolic pathway of reutilization consists of L-fucose kinase and / or GDP-L-fucose pyrophosphorylase. The pool of GDP-mannose is increased by up-regulation of genes encoding GDP-D-mannose pyrophosphorylase, phosphomannose isomerase and / or phosphomannomutase; and / or genes encoding mannokinase and GDP-D-mannose pyrophosphorylase. The pool of UDP-galactose is increased by up-regulation of genes encoding galactokinase and / or galactose-1-phosphate-uridyltransferase and / or UDP-galactose-4-epimerase and / or UDP-galactose / glucose pyrophosphorylase and / or synthase lactose and / or lactose phosphorylase and / or sucrose phosphorylase. The pool of UDP-glucose is increased by up-regulation of genes encoding glucokinase and / or UDP-glucose pyrophosphorylase and / or sucrose phosphorylase and / or phosphoglucomutase and / or sucrose synthase. The CMP-sialic acid pool is increased by up-regulation of genes encoding L-glutamine: D-fructose-6-phosphate aminotransferase and / or phosphoglucosamine mutase and / or glucosamine-1-phosphate acetyltransferase and / or N-acetylglucosamine -1-phosphate uridyltransferase, and / or UDR-N-acetylglucosamine-2-epimerase, and / or N-acetylneuraminic acid synthase, and / or cytidine-5'-monophosphate-N-acetylneuraminate synthetase. The pool of UDP-N-acetylglucosamine increases due to upregulation of genes encoding L-glutamine: D-fructose-6-phosphate aminotransferase, and / or phosphoglucosamine mutase, and / or glucosamine-1-phosphate acetyltransferase, and / or N- acetylglucosamine-1-phosphate-uridyltransferase and / or glucosamine-6-phosphate-N-acetyltransferase and / or phosphoacetylglucosamine mutase and / or UDP-N-acetylglucosamine pyrophosphorylase. The UDP-N-acetylmannosamine pool is increased by up-regulation of genes mediating an increase in the UDP-N-acetylglucosamine pool and / or genes encoding UDP-N-acetylglucosamine-2-epimerase. The UDP-N-acetylglucosamine pool is increased by up-regulation of genes mediating an increase in the UDP-N-acetylglucosamine pool and / or genes encoding UDP-N-acetylglucosamine C4 epimerase. The UDP-glucuronic acid pool is increased by up-regulation of genes mediating an increase in the UDP-glucose pool and / or genes encoding UDP-glucose dehydrogenase. The pool of UDP-xylose is increased by up-regulation of genes mediating an increase in the pool of UDP-glucuronic acid and / or genes encoding UDP-D-xylose synthase. The pool of UDP-galacturonic acid increases due to upregulation of genes mediating an increase in the pool of UDP-glucuronic acid and / or genes encoding UDP-D-glucuronic acid-4-epimerase. The UDP-arabinose pool is increased by up-regulation of genes mediating an increase in the UDP-glucuronic acid pool and / or genes encoding UDP-D-xylose-4-epimerase and / or arabinose kinase and / or UDP-L-arabinose pyrophosphorylase ... The pool of dTDP-rhamnose increases due to up-regulation of genes encoding dTDP-glucose pyrophosphorylase and / or dTDP-glucose-4,6-dehydratase, and / or dTDP-4-dehydroramnose-3,5-epimerase, and / or dTDP- 4-dehydroramnose reductase and / or glucose-1-phosphate thymidyl transferase and / or nucleotide rhamnose synthase.

The term "pool" further refers to the concentrations of metabolites that naturally occur in the wild-type organism, for example, the concentration of a pool of sugar found in nucleotides. The term "enlarged pool" refers to a significantly increased concentration of a specified pool of metabolite as compared to the pool of a metabolite in a wild type organism.

The term "gene upregulation" refers to each genetic modification that results in increased gene expression and / or activity of the gene product. Said genetic modification is a modification of the promoter, in the untranslated region, of the ribosome binding site, coding sequence, gene location, intron / exon structure, and / or transcription terminator, which results in said increase in expression and / or activity.

In addition, the present invention describes genetically modified organisms that are capable of transferring these sugars in nucleotides into mono-, di- or oligosaccharides such as galactose, lactose, lacto-N-biose, lacto-N-triose, lacto-N-tetraose , lacto-N-neotetraose, globotriose, 2'-fucosyllactose, 3-fucosyllactose, 3-sialyllactose, 6-sialyllactose, human milk oligosaccharides, heparosans, chitosans, nodule formation factors (Nod factors), glycolipids and / or aglycones but not limited to, by the enzyme glycosyltransferase.

The present invention also describes organisms that are not killed by lactose and which can additionally modify said lactose with enzymes such as carbohydrate hydrolases, carbohydrate transferases, carbohydrate synthase, acetylase, acyltransferase, carbohydrate phosphatase, polysaccharide lyase, kinase, pyruvylase (pyruvylases ) and / or sulfotransferase, but not limited to them.

The present invention further describes organisms that are not killed by lactose and that no longer degrade lactose as a result of the lactose hydrolase gene in these organisms becoming less functional or non-functional.

The term "genes that become less functional or non-functional" refers to techniques well known to those skilled in the art, such as the use of siRNA, interfering RNA, microRNA, asRNA, mutated genes, gene knockout, transposon mutagenesis, CrispR / CAS, etc. which are used to alter genes in such a way that they have a lower ability (i.e., statistically significant "less ability" compared to a functional wild-type gene) or completely lose the ability (for example, knocked out genes) to produce functionally active end products ... The term "(gene) knockout" thus refers to a gene that becomes non-functional. The term "deleted gene" or "gene deletion" also refers to a gene that becomes non-functional (2, 4-9, 22, 27, 30, 43, 45, 46, 51, 65, 74).

The present invention describes organisms in which genes are introduced / knocked out / upregulated to produce the bioproducts described above. Such genes can be found using genetic databases such as, for example, genbank, but not limited to, or protein databases, such as, for example, uniprot, but not limited to, or enzyme databases such as, for example, a database of these Brenda enzymes, but not limited to this (16, 23, 38), and metabolic pathways for the synthesis of bioproducts are described in such databases as, for example, KEGG, Biocyc, Metacyc (20, 44, 47), but not limited to them. The metabolic pathways for the synthesis of some bioproducts described above can be established using several mathematical tools described in the art (35, 54, 57, 76).

Examples of

1. Materials and methods for working with Escherichia coli

Strains and plasmids

Escherichia coli MG1655 [λ ^- , F ^- , rph-1] and JM109 were obtained from the Netherlands Culture Collection of Bacteria (NCCB). All mutant strains were created using the Datsenko & Wanner method (27).

Wednesday

Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco), and 0.5% sodium chloride (VWR, Leuven, Belgium). Shake flask media contained 2 g / L NH ₄ Cl, 5 g / L (NH ₄ ) ₂ SO ₄ , 2.993 g / L KH ₂ PO ₄ , 7.315 g / L K ₂ HPO ₄ , 8.372 g / L MOPS, 0 , 5 g / L NaCl, 0.5 g / L MgSO ₄ ⋅7H ₂ O, 15 g / L glycerin (unless otherwise specified), 1 ml / L vitamin solution, 100 μL / L molybdate solution and 1 ml / L selenium solution. The pH of the medium was adjusted to 7 with 1M KOH.

The vitamin solution consisted of 3.6 g / l FeCl ₂ ⋅4H ₂ O, 5 g / l CaCl ₂ ⋅2H ₂ O, 1.3 g / l MnCl ₂ ⋅2H ₂ O, 0.38 g / l CuCl ₂ ⋅ 2H ₂ O, 0.5 g / l CoCl ₂ ⋅6H ₂ O, 0.94 g / l ZnCl ₂ , 0.0311 g / l H ₃ VO ₄ , 0.4 g / l Na ₂ EDTA⋅2H ₂ O and 1.01 g / L thiamine ⋅ HCl. The molybdate solution contained 0.967 g / l Na ₂ MoO ₄ ⋅ 2H ₂ O. The selenium solution contained 42 g / l SeO ₂ .

The minimum fermentation medium contained 6.75 g / L NH ₄ Cl, 1.25 g / L (NH ₄ ) ₂ SO ₄ , 1.15 g / L KH ₂ PO ₄ , 0.5 g / L NaCl, 0, 5 g / l MgSO ₄ ⋅7H ₂ O, 30 g / l lactose and 20 g / l sucrose (or other concentrations, if otherwise indicated), 1 ml / l vitamin solution, 100 μl / l molybdate solution and 1 ml / l selenium solution of the same composition as described above.

Cultivation conditions

A pre-culture from one colony on a plate with LB medium was incubated in 5 ml of LB medium for 8 hours at 37 ° C on an orbital shaker at 200 rpm. From this culture, 2 ml was transferred to 100 ml minimal medium in a 500 ml shake flask and incubated for 16 hours at 37 ° C. on an orbital shaker at 200 rpm. The 4% inoculum was used in a 2 or 5 L Biostat B Plus culture vessel with a working volume of 1.5 L or 4 L (Sartorius Stedim Biotech, Melsungen, Germany). The cultivation conditions were as follows: 37 ° C, stirring at 800 rpm and a gas flow rate of 1.5 L / min. Aerobic conditions were maintained by bubbling air. The pH was maintained at 7 with 0.5 M H ₂ SO ₄ and 35% M ammonium solution. The outlet gas was cooled to 4 ° C using an outlet gas cooler (Frigomix 1000, Sartorius Stedim Biotech, Melsungen, Germany). When foam was formed during fermentation, a 10% solution of an organosilicon antifoam agent (BDH 331512K, VWR Int Ltd., Poole, England) was added (approximately 10 μl). The off-gas was analyzed using an EL3020 off-gas analyzer (ABB Automation GmbH, 60488 Frankfurt am Main, Germany).

All data were recorded using the Sartorius MFCS / win system, version 3.0 (Sartorius Stedim Biotech, Melsungen, Germany).

Sampling procedure

Inside the bioreactor there is a collecting tube (BD Spinal Needle, 1.2 × 152 mm (BDMedical Systems, Franklin Lakes, NJ - USA), which is connected to the reactor port, and outside it is connected to a flexible Masterflex-14 tube (Cole-Parmer, Antwerp, Belgium), terminating in a collection port with a sampling baffle.On the other hand, this collection port is connected back to the reaction vessel using a flexible Masterflex-16 tubing.This system is called a fast sampling loop. During sampling, the reaction broth is pumped through a loop for It was calculated that at a flow rate of 150 ml / min, the reaction broth reaches the collecting port in 0.04 seconds and returns to the reactor in 3.2 seconds.At a pO ₂ level of 50%, the oxygen content of the liquid is approximately 3 mg / l at 37 ° C. The pO ₂ level should never fall below 20% to avoid microaerobic conditions. Thus, 1.8 m can be absorbed during the passage through the collecting loop. g / l oxygen. If we assume that the oxygen uptake rate is 0.4 g oxygen / g biomass / h (maximum oxygen uptake rate at μ _max ), then for 5 g / l biomass the oxygen uptake rate will be 2 g / l / h or 0.56 mg / l / s, taking into account 3.2 s (residence time of the biomass in the loop), oxygen uptake will be 1.8 mg / l.

To suppress cell metabolism during sampling, the reaction broth was aspirated from the collection port using a syringe filled with 62 g stainless steel beads pre-cooled to -20 ° C to immediately cool 5 ml of broth to 4 ° C. After sampling, the samples were immediately centrifuged with refrigeration (15000 g, 5 min, 4 ° C). During periodic experiments, samples for measuring OD _600nm were taken using a quick sampling loop and a chilled steel ball sampling method. Analysis methods

The density of the culture cells was continuously monitored by measuring the optical density at 600 nm (spectrophotometer Uvikom 922, BRS, Brussels, Belgium). The dry cell mass was obtained by centrifugation (15 min, 5000 g, GSA rotor, Sorvall RC-5B, Goffin Meyvis, Capellen, Belgium) 20 g of reaction broth in pre-dried and weighed Falcon tubes. Then the precipitate was washed once with 20 ml of physiological solution (9 g / L NaCl) and dried at 70 ° C to constant weight. To be able to convert the OD _600nm measurements into biomass concentration values, a correlation curve was _drawn between the OD 600nm and the biomass concentration. Concentrations of glucose and organic acids were determined using a Varian Prostar HPLC system (Varian, Sint-Cateleine-Waver, Belgium) using an Aminex HPX-87H column (Bio-Rad, Ecke, Belgium) heated to 65 ° C, equipped with a 1 cm, and using as a mobile phase 5 mm H ₂ SO ₄ (0.6 ml / min). A dual-wavelength UV-VIS (210 nm and 265 nm) detector (Varian Prostar 325) and a differential refractometric detector (Merck LaChrom L-7490, Merck, Leuven, Belgium) were used for peak detection. Peaks can be identified by dividing the absorbance value for the peak at 265 by the absorbance value for the peak at 210 nm. The division results are a constant value characteristic of a particular compound (Lambert-Beer formula).

Glucose, fructose, sucrose, oligosaccharides and glucose-1-phosphate were measured by HPLC using a Hypercarb column and detected using an MSMS detector (Antonio et al., 2007; Nielsen et al., 2006).

Genetic ways

The methods used to obtain mutants are described below.

Plasmids were maintained in E. coli DH5α host cells (F ^- , ϕ80dlacZΔM15, Δ (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17 (rk ^- , mk ⁺ ), phoA, supE44, λ ^- , thi-1, gyrA96, relA1).

Plasmids

Plasmids pKD46 (auxiliary plasmid Red, ampicillin resistance), pKD3 (contains the FRT-flanked chloramphenicol resistance gene (cat)), pKD4 (contains the FRT-flanked kanamycin resistance gene (kan)), and pCP20 (expresses FLP recombinase) used to obtain mutant constructs. Plasmid pBluescript (Fermentas, St. Leon-Rot, Germany) was used to obtain derivatives of pKD3 and pKD4 with a library of promoters or alleles carrying a point mutation.

Mutations

The mutations consisted of gene dysfunction (knockout, KO). They were introduced using the concept of Datsenko and Warmer (27).

Transformants containing the auxiliary plasmid Red were grown in 10 ml of LB medium with ampicillin (100 mg / L) and L-arabinose (10 mM) at 30 ° C to an OD _{600 nm} of 0.6. To obtain electrocompetent cells, the cells were washed first with 50 ml of ice water, then with 1 ml of ice water. Then the cells were resuspended in 50 μl of ice water. Electroporation was performed using 50 μl of cells, 10-100 ng of linear double-stranded DNA product and Gene Pulser ™ (BioRad) (600 Ω, 25 μF and 250 volts).

After electroporation, the cells were added to 1 ml of LB medium and incubated for 1 h at 37 ° C, after which they were plated on LB agar containing 25 mg / L chloramphenicol or 50 mg / L kanamycin to select transformants resistant to antibiotic. The presence of mutations in the selected mutants was confirmed by PCR with primers to the regions of the sequence located above and below the modified region, then the selected mutants were grown on LB agar at 42 ° C so that they lost the auxiliary plasmid. The mutants were tested for sensitivity to ampicillin.

Linear double stranded DNA

Linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivatives as templates. Part of the sequence of the primers used was complementary to the template, and the other part was complementary to the region on the chromosomal DNA where recombination is to take place. For KO, a region of homology 50 nucleotides before the start codon of the target gene and a region of homology 50 nucleotides after the stop codon of the target gene were constructed. For KI, it was necessary to preserve the transcription start point (+1). PCR products were purified after PCR, digested with DpnI, purified a second time in agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0).

Removal of the antibiotic resistance gene. The selected mutants (resistant to chloramphenicol or kanamycin) were transformed with the plasmid pCP20, which is a plasmid with resistance to ampicillin and chloramphenicol, which has temperature-sensitive replication and thermal induction of FLP synthesis. Ampicillin-resistant transformants were selected at 30 ° C, after which several colonies were purified on LB at 42 ° C and then checked if they had lost all antibiotic resistance and the auxiliary plasmid FLP. Knockouts and gene insertions were verified using control primers (Fw / Rv-gene-out).

Transformation

Plasmids were transformed into CaCl ₂ competent cells by the simplified Hanahan technique (42) or by electroporation as described above.

2. Materials and methods for working with yeast

2.1. Strains

Saccharomyces cerevisiae BY4742 were obtained from the Euroscarf culture collection. All mutant strains were created by homologous recombination or by transformation with a plasmid using the Gietz method (40). Kluyveromyces marxianus lactis were obtained from the culture collection of the Laboratory of Industrial Biotechnology and Biocatalysis.

2.2. Wednesday

The strains were grown on a synthetic medium with a well-defined composition for yeast with a complete additive mixture (Synthetic Defined yeast medium with Complete Supplement Mixture, SD CSM) or CSM drop-out (SD CSM-Ura) containing 6.7 g L ^-1 base nitrogen agar for yeast without amino acids (YNB w / o AA, Difco), 20 g L ^-1 agar (Difco) (solid cultures), 22 g L ^-1 glucose monohydrate or 20 g L ^-1 lactose and 0, 79 g ^{l -1} CSM or 0.77 g ^{l -1} CSM-Ura (MP Biomedicals).

2.3. Cultivation conditions

Yeast cultures were first inoculated into 5 ml of a suitable medium and incubated overnight at 30 ° C and 200 rpm. To increase the volume of the culture, 2% (or more) of the pre-culture was inoculated into 50-200 ml of medium. These cultures were incubated again at 30 ° C and 200 rpm.

Growth assay experiments were performed in a 96-well plate or Erlenmeyer flask. To obtain single colonies as starting material for experiments on the analysis of growth and production, the strains were plated on plates with selective medium SD CSM and incubated for 2-3 days at 30 ° C. Then one colony was selected and transferred into 5 ml of medium for experiments in an Erlenmeyer flask or in 1 ml of medium for experiments in microtiter plates.

For experiments in an Erlenmeyer flask, pre-cultures were incubated overnight at 30 ° C and 200 rpm and 2% of these pre-cultures were added to 100 ml of medium to start growth assay experiments.

For MTP growth assay experiments, colonies were added to 150 μl of medium and incubated for 24 hours at 30 ° C. After incubation, 2 μl of MTP pre-cultures were added to 150 μl of fresh medium containing MTP. OD was measured every fifteen minutes for 48 hours using the Infinite® 200 Pro Tecan.

2.4. Sampling procedure

Samples for measuring OD, cell fraction (0.2 ml) and supernatant fraction (1 ml) of the culture were taken every two hours until the stationary phase was reached and every couple of hours during the stationary phase. A 1 ml sample was first centrifuged (11), after which the cell pellet and supernatant were separated and stored separately at -20 ° C. The supernatant was stored for analysis of extracellular products, and the cell pellet was used for analysis of intracellular metabolites.

2.5. Analysis methods

The density of the culture cells was constantly monitored by measuring the optical density at 600 nm (Uvikom 922 spectrophotometer, BRS, Brussels, Belgium) or using a Biochrom Anthos Zenyth 340 microtiter plate reader. The dry mass of cells was obtained by centrifugation (15 min, 5000g, GSA rotor, Sorvall RC -5B, Goffin Meyvis, Capellen, Belgium) 20 g reaction broth in pre-dried and weighed Falcon tubes. Then the precipitate was washed once with 20 ml of physiological solution (9 g / L NaCl) and dried at 70 ° C to constant weight. To be able to convert the OD _600nm measurements into biomass concentration values, a correlation curve was _drawn between the OD 600nm and the biomass concentration. Concentrations of glucose and organic acids were determined using a Varian Prostar HPLC system (Varian, Sint-Cateleine-Waver, Belgium) using an Aminex HPX-87H column (Bio-Rad, Ecke, Belgium) heated to 65 ° C, equipped with a 1 cm and using as a mobile phase 5 mm H ₂ SO ₄ (0.6 ml / min). A two-wavelength UV-VIS detector (210 nm and 265 nm) (Varian Prostar 325) and a differential refractometric detector (Merck LaChrom L-7490, Merck, Leuven, Belgium) were used to detect the peak. Peaks can be identified by dividing the absorbance value for the peak at 265 by the absorbance value for the peak at 210 nm. The division results are a constant value characteristic of a particular compound (Lambert-Beer formula).

Glucose, fructose, sucrose, oligosaccharides and glucose-1-phosphate were measured by HPLC using a Hypercarb column and detected using an MSMS detector (Antonio et al., 2007; Nielsen et al., 2006).

2.6. Genetic ways

The methods used to obtain mutants are described below.

Plasmids were maintained in E. coli DH5α host cells (F ^- , ϕ80dlacZΔM15, Δ (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17 (rk ^- , mk ⁺ ), phoA, supE44, λ ^- , thi-1, gyrA96, relAX).

Plasmids

The yeast expression plasmid p2a_2μ_Lac4, obtained from the Laboratory of Industrial Biotechnology and Biocatalysis, was used to enable the growth of Saccharomyces on lactose as the sole source of C. This plasmid contains an ampicillin resistance gene and a bacterial origin of replication to allow selection and maintenance of the plasmid in E . coli. The plasmid also contains 2μ ori yeast and the Ura3 selectable marker to allow selection and maintenance of the plasmid in yeast. Finally, the plasmid contains the β-galactosidase expression cassette (SEQ ID 9, Figure 15).

Mutations

The mutations consisted of the introduction of the plasmid p2a_2μ_Lac4 (as described above) and the insertion of the gene (KI) (KI at the rDNA locus) using double-stranded linear DNA (as described above). Transformants were plated on SD CSM-Ura medium after transformation with plasmid DNA or on SD CSM-Ura medium with lactose as the only C source after transformation with double-stranded linear DNA. The presence of plasmids in the selected mutants was confirmed by PCR with primers corresponding to p2a_2μ_Lac4. The presence of a genomic insert in the selected mutants was confirmed by PCR with primers to the regions above and below the modified region, and also confirmed by sequencing (performed at LGC Genomics (LGC group, Germany)).

Linear double stranded DNA

Linear ds-DNA amplicons were obtained by PCR using pJet_KI_p1_Lac12_t @ rDNA or pJet_KI_p2_Lac12_t @ rDNA plasmids as templates. These plasmids contain 2500 bp homology regions (HR1 (SEQ ID 10, Figure 16) and HR2 (SEQ ID 11, Figure 17)) flanking SEQ ID 7 and SEQ ID 8, respectively, inserted at the multiple cloning site of the pJET cloning vector (Thermoscientific). The primers used are homologous to the 5 'end of HR1 (forward primer) and the 3' end of HR2 (reverse primer). Before transformation, PCR products were purified after PCR.

Transformation

Plasmids and linear double-stranded DNA were transformed using the Gietz method (40).

3. Results

Example 1. Insertion of lactose permease lacYE. coli to the agp locus.

The first strain MG1655 ΔlacY was created according to the method of Datsenko and Wanner, as described above. For this MG1655 was transformed with the plasmid pKD46 and linear DNA was constructed from the basic plasmids pKD3 and pKD4 with flanked regions of homology to the lacY gene. Successful recombinants were then screened with appropriate antibiotics. To ensure that this strain is not capable of absorbing lactose, the strain was grown on minimal medium containing only lactose as a carbon source. No growth was observed during this experiment, therefore, this cell can no longer transport lactose across its membrane.

To further obtain a synthetic expression system, a synthetic promoter and RBS were synthesized in combination with the lacY gene (ordered from IDT and Geneart). This sequence is shown in Figure 3. This sequence was also introduced into the genome at the agp gene locus following the adapted technique of Datsenko and Wanner. Briefly, the lactose permease construct was first assembled with a screening cassette from plasmid pKD3, resulting in a new plasmid pCX_lacY-kan. From this plasmid, linear DNA can be amplified by PCR with homology to the agp genomic region. This resulting linear DNA can then be transformed into MG1655 ΔlacY E coli, in which the plasmid pKD46 is present. As a result, the lactose transporter expression cassette is recombined into the genome and an organism expressing the lactose permease, MG1655ΔlacYΔagp :: lacY _{synthetic, is formed} . To confirm the restoration of the ability to grow on lactose, this strain was grown on minimal medium containing lactose, as described above. The ability to grow on lactose was fully restored, and the growth rate was similar to that of the wild-type strain.

Example 2. Influence of lactose on wild-type E. coli strain and mutant E. coli strain, which does not undergo death under the influence of lactose

The shake flask experiment, as described in the materials and methods, was performed on MG1655 wild type and MG1655ΔlacYΔagp :: lacY _synthetic . These strains were grown in a shake flask in medium with glycerol (15 g / L glycerol, as described in materials and methods), and in the middle of the exponential phase (at approximately OD 0.8), lactose was added to the strains (200 g / L concentrated solution was added to a final concentration of 10 g / l). As can be seen in Figure 1 and Figure 2, the mutant strain is not killed by lactose.

Example 3. Use of Translational Binding or Translational Sensors to Confirm Expression of a Lactose Transporter

Since a strain with a completely knockout gene of lactose permease will also not undergo death under the action of lactose, and the goal is to obtain a functional, active, expressed lactose permease, it is necessary to screen for the expression of lactose permease. For this purpose, variants of promoter sequences, ribosome binding sites, Kozak sequences, variants with altered codon frequency and variants of transcription terminators can be created. However, with these sequence variants, null expression constructs can be produced, resulting in mutant strains lacking the lactose transporter. Therefore, a system must be provided for detecting the expression of a lactose transporter, preferably with a reporter gene that can be easily screened for, such as lacZ, fluorescent protein genes or antibiotic resistance genes.

Creation of a translationally coupled system that re-initiates translation of a reporter gene to detect lactose transporter expression

The two genes can be translationally linked by introducing a translation re-initiation region located 3 'from the target gene and 5' from the reporter gene. Translation can be re-initiated using several codons such as AUG, UUG, GUG, or AUA (63). The sequence of such a construct, which translationally links the lactose permease gene and the lacZ gene, is shown in Figure 4. To generate this sequence, the lacY and lacZ sequences are amplified from the E. coli genome using primers with golden gate restriction sites (BsaI, obtained from NEB). The intergenic region providing translational binding can be ordered from any gene synthesis company such as IDT or Geneart. All parts are further assembled by the Golden Gate method as described by Engler et al. (2013) (36), into the plasmid pUC54 together with the promoter and RBS sequence, as shown in Figure 3 (for E. coli), or into the plasmid pGK12, which is replicated in Bacillus sp. and contains a promoter and a Bacillus ribosome binding site.

Creation of a translationally linked system that initiates translation of a reporter gene for detecting the expression of a lactose transporter by opening a loop at the ribosome binding site

A second method of screening for expression based on translational binding is described by Mendez-Perez et al. (2012) (58). This method has been adapted for screening for expression of lactose permease with a chloramphenicol resistance reporter gene. In this case, the lactose transporter lacY is linked via a HIS-tag and a ribosome binding site to the chloramphenicol resistance reporter gene. Sequence parts for this construct are also ordered from IDT or Geneart and assembled using the Golden gate assembly method. The resulting sequence is shown in Figure 5.

Creation of a translationally linked system linking the yeast lactose transporter with the reporter gene

Although yeast lacks cistrons, it is possible to screen for expression by translational binding through viral ribosomal intrinsic entry sites (so-called IRES sequences) (56). An example of such a sequence is the T2A sequence (10), which provides completely independent (meaning that the proteins are not a fusion protein), but linked translation of the two proteins in the cistron. This means that if the last cistron protein is expressed, then the first protein is also expressed.

In yeast such as Kluyveromyces marxianus, the lactose permease gene can be used to transport lactose into the cell. This gene can be linked via the T2A sequence to the aph 1 gene encoding geneticin resistance. This sequence is created in the same way as described above. The final sequence is shown in Figure 6.

Creation of a system associated with an aptamer, with the help of which the aptamer is introduced into messenger RNA of lactose permease

Expression of lactose permease can also be detected at the messenger RNA level. For this, the aptamer binding (Z) -4- (3,5-difluoro-4-hydroxybenzylidene) -1,2-dimethyl-1H-imidazol-5 (4H) -one is cloned after the coding sequence of lactose permease, as shown in Figure 7. Expression of this construct is modulated as described in Example 6. After the completion of cell growth, (Z) -4- (3,5-difluoro-4-hydroxybenzylidene) -1,2-dimethyl-1H-imidazole is added to the medium -5 (4H) -one as described by Pothoulakis et al (2013) (64) and mutant strains expressing lactose permease are selected on a fluorescence activated cell sorter (FACS).

Example 4. Detection of expression of a lactose transporter translationally linked to the chloramphenicol resistance gene

Two strains were designed in which lactose permease was removed (knocked out) from the genome. Both strains were transformed with plasmid pSC101, contains the kanamycin resistance gene, with the difference that one of the plasmids contained a constitutively expressible lactose permease as described in Example 1 and 2, whereby were obtained comparing the strain MG1655ΔlacY pSC101_kan and strain MG1655ΔlacY pSC101_kan_ lacY _synthetic _cat with translational binding of lacY_cat as described in Example 4 and Figure 5. Both strains were grown in minimal medium as described above at different chloramphenicol concentrations (between 0 and 30 mg / L). Figures 8 and 9 show that after 48 and 92 hours, the growth of the reference strain is inhibited at a lower concentration of chloramphenicol compared to the translationally binding mutant strain lacY_cat, suggesting that such a system is an excellent option for screening genetic constructs expressing lactose. permease.

Example 5. Method of screening for the presence of mutants expressing lactose permease and not subject to death by lactose

As in Example 2, a mixture of two strains resistant to chloramphenicol was grown, where one strain does not die under the action of lactose and expresses lactose permease translationally associated with chloramphenicol, and the second strain has a chloramphenicol resistance cassette, but contains the natural expression system of lactose permease. Both strains were grown in medium as described in Example 2, and lactose was added in the middle of the exponential phase, as shown in Example 2. The mutant strain that did not die with lactose continued to grow, while the other strain that was susceptible to death with lactose stopped growing. At the end of the exponential phase, 0.1 ml of this culture was inoculated into a second shake flask with the same medium as described above. Again, at an OD of 0.8, lactose was added, which caused the growth of the strain susceptible to death by lactose to be stopped and the accumulation of the mutant strain not subject to death by lactose. After 5-fold repetition of this procedure, the proportion of the mutant strain that did not undergo death under the action of lactose was 99%, which made it easy to isolate it from the culture.

Example 6. Creation of mutant lactose permease expression cassettes

Sequence variants of promoters, ribosome binding sites, Kozak sequences, transcription terminators, and variants of the lactose permease gene (with different codon frequency) were ordered from companies offering gene synthesis services, for example, IDT, Geneart, Genscript, Gen9, and others. The design of the bacterial promoter library is based on the consensus sequence of bacterial promoters with two conserved regions at -10 and -35 bp from the transcription start point. The bases between these conserved regions, before and after them were then randomly changed to A, T, G or C, as a result of which promoters with different expression strengths were obtained. Alternatively, the conserved regions were altered and the surrounding sequences retained, resulting in promoters of different strengths as well. These mixtures of sequences were then cloned (either by Gibson assembly, or by Golden Gate assembly, or by Cliva assembly, or LCR, or by restriction and ligation) (25, 36, 50, 79) before translationally coupled lactose permease, resulting in a library of lactose permease expression cassettes that can be screened using the protocol described in Example 5.

A library of eukaryotic promoters was created based on the core promoter (13). For Saccharomyces cerevisiae, this promoter may be the heterologous TEF promoter pTEF1, which is amplified by UAS sequences and modified by mutations to alter the strength of the promoter (12). Such a promoter was ordered and cloned as described above prior to translationally linked lactose permease and the final construct was transformed into yeast, such as Saccharomyces cerevisiae, using a plasmid or by chromosome integration.

The untranslated region in prokaryotes consists of the ribosome binding site, while in eukaryotes it consists of the Kozak sequence. Both sequences were randomized and cloned before the coding sequence as described above, resulting in a library of expression cassettes with different translational efficiencies. Randomization can be rationalized with tools such as the RBS calculator, which calculates the correlation of sequence translation efficiency and reduces the number of variants that must be included in the library (67).

The codon frequency was changed by changing the coding sequence of the gene without changing the amino acid sequence of the protein. This is called codon adaptation. The frequency of codon use throughout the sequence was changed in such a way that more or less rare codons were introduced into certain regions, which led to a change in the expression efficiency and folding efficiency. Permease, the sequence of which contains only rare codons (determined for the organism using the codon frequency database (61)), is characterized by a low translation rate compared to permease with a fully codon-optimized sequence (contains only those codons that are often found in the target organism). In addition, the first codons of the coding sequence also affect the efficiency of the Kozak sequence or the ribosome binding site (62, 69, 78).

The transcription terminator region was altered using endogenous or exogenous transcription terminator sequences from the database (18, 26). These transcription terminators were also cloned in a manner similar to that described above.

Example 7. Enrichment of expression cassettes that express lactose permease and do not lead to death by lactose

Expression cassettes were created in accordance with Examples 3 and 7. As a result, a library of expression cassettes of lactose permease was obtained. Expression cassettes that resulted in the expression of lactose permease were selected according to the methods of Examples 3 and 4. Expression cassettes expressing lactose permease that did not result in death by lactose were selected according to the methods described in Examples 2 and 5. Selected expression cassettes were also analyzed using sequencing and the method described in Example 2.

Example 8. Enzymatic production of 2-fucosyllactose by bacteria E. coli with fucosyltransferase from Helicobacter pylori

A mutant strain in which the genes lacZ, glgC, agp, pfkA, pfkB, pgi, arcA, iclR, wcaJ are knocked out and lacY is constitutively expressed, as described in Example 1 and Example 2, to ensure expression under all cultivation conditions, was then transformed with fucosyltransfera Helicobacter pylori and sucrose phosphorylase from Bifidobacterium adolescentis, which are also expressed constitutively. Constitutive promoters were selected from the promoter library described by De Mey et al., 2007. This strain was cultured in medium as described in materials and methods, but with 30 g / L sucrose and 50 g / L lactose. As a result, up to 1.5 g / l of 2'-fucosyllactose was obtained

Example 9. Production of 2-fucosyllactose by E. coli Bacteria with Batch Feeding

Using genetic engineering methods according to the methods described above, a mutant strain with the genotype was created:

ΔlacZYAΔglgCΔagpΔpgiΔpfkA-P22-baSPΔpfkBΔarcAΔiclR :: slΔwcaJAΔlonΔadhE-P14-frk + pCXP14-FT_H. pylori (vector with sequence SEQ ID No. 6, see Figure 10). The expression of lactose permease was altered as described in Example 1 and Example 2. The P22 and P14 promoters were obtained from a promoter library constructed by De Mey et al. (29), and they were cloned by analogy with the method described by Aerts et al. (one). ":: sl" means "scarless" gene deletion, that is, without the FRT site that remains on the chromosome.

This strain was cultured in a bioreactor, as described above in materials and methods, in a mineral medium with 30 g / l sucrose and 50 g / l lactose. After the batch phase, 500 g / L sucrose, 50 g / L lactose and 1 g / L magnesium sulfate heptahydrate were added to the bioreactor. As a result, 27.5 g / L of fucosyllactose was accumulated in the supernatant.

Example 10. Production of lacto-N-triose by bacteria E. coli

The mutant strain was constructed by genetic engineering methods according to the methods described above, for the expression of UDP-N-acetylglucosamine transferase, sucrose phosphorylase, L-glutamine: D-fructose-6-phosphate aminotransferase and glucosamine uridyltransferase using the plasmid pBR322, in which each the gene was expressed under the control of a constitutive promoter from the De Mey et al. promoter library. (29), and contained a selective marker beta-lactamase. This vector was transformed into a mutant E. Coli strain having the genotype ΔlacZYAΔglgCΔagp :: P14-frk- ¥ 22-BaSPΔpgiΔpfkAΔpfkBΔnagABCDEΔmanAΔnanATEKΔmanXYZ constitutively expressing lactose permease as described above. This strain was cultured in a shake flask as described above with lactose and sucrose as carbon sources with or without added glycerol. This producer host was not killed by lactose and produced 62.5 and 55.3 mg / L lacto-N-triose, respectively, from the added lactose.

Example 11. Construction of the lactose permease of the yeast K. marxianus (pi) introduced into the rDNA locus

First, the wild-type Saccharomyces cerevisiae BY4742 strain was transformed with the plasmid p2a_2μ_Lac4. Transformation was carried out using a total of 4 μg of plasmid according to protocol (40). Transformed yeast cells were plated on SD-CSM drop-out plates (no uracil). After two days, growth was observed on the plates and yeast colonies were tested for the presence of the desired plasmid. Colony selection PCR was performed for all 33 colonies. All colonies tested contained the p2a_2μ_Lac4 plasmid. Colony 5 was selected for further use. This colony was transformed with 2 μg of double-stranded linear DNA obtained from pJet_KI_p1_Lac12_t @ rDNA. The transformed cells were grown on SD-CSM-Ura plates with lactose as the sole source of C. Three days after transformation, several colonies had grown enough to be tested for the presence of the Lac12 expression cassette in Saccharomyces rDNA. For all colonies, PCR was performed to select the colonies. All colonies tested contained the Lac12 expression cassette. Colony 6 {Saccharomyces KI_p1_Lac12_t @ rDNA) was selected for further use.

Example 12. Construction of the lactose permease of the yeast K. marxianus (p2) introduced into the rDNA locus

First, the wild-type Saccharomyces cerevisiae BY4742 strain was transformed with the plasmid p2a_2μ_Lac4. Transformation was carried out using a total of 4 μg of plasmid according to the Gietz protocol (40). Transformed yeast cells were plated on SD-CSM drop-out plates (no uracil). After two days, growth was observed on the plates and yeast colonies were tested for the presence of the desired plasmid. Colony selection PCR was performed for all 33 colonies. All colonies tested contained the p2a_2μ_Lac4 plasmid. Colony 5 was selected for further use. This colony was transformed with 2 μg of double-stranded linear DNA obtained from pJet_KI_p2_Lac12_t @ rDNA. The transformed cells were grown on SD-CSM-Ura plates with lactose as the sole source of C. Three days after transformation, several colonies had grown enough to be tested for the presence of the Lac12 expression cassette in Saccharomyces rDNA. For all colonies, PCR was performed to select the colonies. All colonies tested contained the Lac12 expression cassette. Colony 7 (Saccharomyces KI_p2_Lac12_t @ rDNA) was selected for further use.

Example 13. Growth on lactose of a wild-type yeast strain and 2 mutant yeast strains

The shake flask experiment, as described in materials and methods, was performed on wild type Kluyveromyces, Saccharomyces KI_p1_Lac12_t @ rDNA and Saccharomyces KI_p2_Lac12_t @ rDNA strains. These strains were grown in a shake flask in a medium containing lactose as the only source of C (20 g / L). As can be seen in Table 1, the mutant Saccharomyces strains grow as fast as the wild-type Kluyveromyces marcianus lactis strain, which is known to be capable of rapid growth on lactose (31). Thus, constitutively expressed lactose permease provides a fast and efficient influx of lactose into the yeast cell.

Example 14. Effect of lactose on a wild-type yeast strain and a mutant yeast strain that does not die under the action of lactose

The shake flask experiment, as described in materials and methods, was performed on wild type Kluyveromyces, Saccharomyces KI_p1_Lac12_t @ rDNA and Saccharomyces KI_p2_Lac12_t @ rDNA strains. These strains were grown in a shake flask in a medium with glucose (20 g / L glucose, as described in materials and methods), and in the middle of the exponential growth phase, lactose was added to the strains (200 g / L of concentrated solution was added to a final concentration of 10 g / L ). As can be seen in Figure 11 and Figure 12, the mutant strains are not killed by lactose. Moreover, in Example 13, the presence of a fast and efficient flow of lactose into the interior of these yeast cells was confirmed.

Example 15. Sequencing of lactose permease expression cassettes that do not lead to death by lactose

46 colonies selected by screening as described above were sequenced, resulting in the sequence of SEQ ID Nos. 12 to 57 (Figure 18). These sequences are promoter and RBS variants that are non-lactose-free when expressed in E. coli. It should be noted that a huge number of colonies were selected and were sequenced, therefore, using this screening technique, many more sequences were obtained than shown in Figure 18, and the alternative library methods described above will also lead to sequences. different from those shown in Figure 18.

Example 16. Determination of μ _MAX for mutant strains with a lactose permease expression cassette

All strains were taken from either LB agar plates on which they were grown or from cryovials and inoculated into 5.0 ml of Luria broth medium (10 g tryptone; 5 g yeast extract; 10 g NaCl). After growing overnight at 37 ° C, 1 ml of this pre-culture was added to a 500 ml shake flask containing 100 ml of lactose minimal medium (2.0 g / L NH ₄ Cl; 5.0 g / L; (NH ₄ ) ₂ SO ₄ ; 3.0 g / l KH ₂ PO ₄ , 7.3 g / l K ₂ HPO ₄ ; 8.4 g / l MOPS; 0.5 g / l MgSO ₄ × 7H ₂ O; 0, 5 g / l NaCl; 10 g / l lactose; 0.4 mg / l Na ₂ EDTA × 2H ₂ O; 0.03 mg / l H ₃ VO ₃ ; 1.01 mg / l thiamine HCl; 0.94 mg / l ZnCl ₂ ; 0.5 mg / l CoCl ₂ × 6H ₂ O; 0.38 mg / l CuCl ₂ × 2H ₂ O; 1.59 mg / l MnCl ₂ × 4H ₂ O; 3.6 mg / l CaCl ₂ and 0.096 mg / l Na ₂ MoO ₄ × 2H ₂ O); pH 7.0. After growing overnight at 37 ° C, both pre-cultures were diluted with lactose minimal medium to an OD ₆₀₀ of 0.050. The suspension was then transferred to a 96-well MTP plate (n = 32), covered with Easyseal film. OD measurements were carried out every 10 minutes for 24 hours using Infinite M200 pro (TECAN) under the following conditions: temperature: 37 ° C +/- 0.5; shaking 597 seconds; shaking amplitude 2 mm; 280 rpm; wavelength OD ₆₀₀ ; Flash # 10; flash time 150 ms).

Example 17 Characterization of lactose permease expression cassettes

One way to reduce or eliminate death by lactose is to significantly reduce or eliminate the activity of lactose permease (see Example 16). But from the point of view of the production of biological products based on lactose or galactose, it is necessary that a lot of lactose enter the cell. Here we confirm that the influx of lactose into the cell in mutant strains resistant to death under the action of lactose is comparable to or even exceeds the influx of lactose into the cell in a wild-type organism undergoing death under the action of lactose. For this purpose, new lactose permease expression cassettes, still expressing beta-galactosidase, were introduced into the MG1655ΔlacY strain. The growth rate of these new strains is indicative of the influx of lactose into the cell, since any strain with markedly reduced lactose permease expression will have a significantly slower growth rate.

The results of this analysis are shown in Figure 19. Almost all of the strains shown in this figure have a growth rate equal to or faster than the wild-type strain, indicating that the activity and expression of lactose permease for them is equal to or higher than in the wild-type strain, however, unlike the wild-type expression system, these expression cassettes do not result in death by lactose. This method, however, is limited to the expression of the beta-galactosidase gene, which becomes a growth limiting stage. Therefore, the expression of the lactose permease gene was measured using the above-described translationally linked system. The minimum inhibitory concentration (MIC) for chloramphenicol is an indication of the expression of the lactose permease gene, and the MIC was determined for each cassette. The lowest MIC was used as an indicator of wild-type lactose permease expression, which still had the same growth rate as the wild-type strain.

For strains containing cassettes with the lowest MIC and having the same growth rate as the wild-type strain, the MIC is approximately 20 mg / L chloramphenicol. For mutant strains with a slightly lower growth rate, the MIC is in the range between 15 and 20 mg / L, and for mutant strains with the same or higher growth rate, the MIC is in the range between 20 and 80 mg / L. 85% of the sequences belong to the latter category, which means that most of the sequences identified as expression cassettes that do not lead to death by lactose show higher expression of lactose permease, which is contrary to that previously described in the literature.

Example 18 Creation of an expression cassette with the lacIq promoter

By analogy with the procedure described above, the placIQ promoter was cloned upstream of the E. coli lacY gene. The final sequence of this design is shown in Figure 20.

Example 19 Other Promoters Used in the Prior Art Which Lead to Death by Lactose

The placIQ promoter is a promoter that has been used in the art to express lactose permease in E. coli. When using this promoter, lactose is absorbed into the cell. However, the effect of such absorption was not tested. The growth rate of the wild-type strain on lactose does not differ significantly from the growth rate of the strain with the lacIq promoter (μmax is 0.1 and 0.11 h ^-1 , respectively), which indicates approximately equal rates of lactose absorption. Screening for the phenomenon of lactose death in strains with such a lacIQ expression cassette confirmed that this promoter also leads to lactose death (see Figure 21). This confirms the fact that there are certain sequences of promoters that lead to death by lactose, and other sequences that do not lead to death by lactose. Sequence rationalization is not possible, and selection of individual sequences (by trial and error) is painstaking work that requires huge costs, thus, the method described above for identifying expression cassettes that do not lead to death by lactose is the best alternative to the long and expensive work on screening.

Example 20. Production of lacto-N-tetraose by S. cerevisiae yeast

The mutant strains constructed in Examples 11 and 12 were further transformed with β-1,4-galactosyltransferase and β-1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis, which were also constitutively expressed using a standard yeast expression vector such as described by Lee et al. ... (52). The strains were cultured in the medium as described in the materials and methods, but with 20 g / L sucrose and 20 g / L lactose. As a result, up to 30 mg / l of lacto-N-tetraose was obtained.

Example 21. Production of 2-fucosyllactose by the yeast S. cerevisiae

The mutant strains constructed in Examples 11 and 12 were further transformed with GDP-fucose synthase and GDP-mannose-4,6-dehydratase E. coli and fucosyltransferase from Helicobacter pylori, which were also constitutively expressed using a standard yeast expression vector, for example, described by Lee et al. (52). The strains were cultured in the medium as described in the materials and methods, but with 20 g / L sucrose and 20 g / L lactose. As a result, up to 10 mg / l of 2-fucosyllactose was obtained.

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--->

LIST OF SEQUENCES

<110> UNIVERSITY GENT

<120> MUTANT MICROORGANISMS RESISTANT TO DEATH BY LACTOSE

<130> P2013 / 049PCT

<150> EP14193151.9

<151> 2014-11-14

<160> 58

<170> PatentIn version 3.5

<210> 1

<211> 1460

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 1

agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct tgtgtaggct 60

ggagctgctt cgaagttcct atactttcta gagaatagga acttcggaat aggaactaag 120

gaggatattc atatggaccg atatcccggg cggccgctct agaagaagct tgggatccgt 180

cgacctcgaa ttcggaggaa acaaagatgt actatttaaa aaacacaaac ttttggatgt 240

tcggtttatt ctttttcttt tactttttta tcatgggagc ctacttcccg tttttcccga 300

tttggctaca tgacatcaac catatcagca aaagtgatac gggtattatt tttgccgcta 360

tttctctgtt ctcgctatta ttccaaccgc tgtttggtct gctttctgac aaactcgggc 420

tgcgcaaata cctgctgtgg attattaccg gcatgttagt gatgtttgcg ccgttcttta 480

tttttatctt cgggccactg ttacaataca acattttagt aggatcgatt gttggtggta 540

tttatctagg cttttgtttt aacgccggtg cgccagcagt agaggcattt attgagaaag 600

tcagccgtcg cagtaatttc gaatttggtc gcgcgcggat gtttggctgt gttggctggg 660

cgctgtgtgc ctcgattgtc ggcatcatgt tcaccatcaa taatcagttt gttttctggc 720

tgggctctgg ctgtgcactc atcctcgccg ttttactctt tttcgccaaa acggatgcgc 780

cctcttctgc cacggttgcc aatgcggtag gtgccaacca ttcggcattt agccttaagc 840

tggcactgga actgttcaga cagccaaaac tgtggttttt gtcactgtat gttattggcg 900

tttcctgcac ctacgatgtt tttgaccaac agtttgctaa tttctttact tcgttctttg 960

ctaccggtga acagggtacg cgggtatttg gctacgtaac gacaatgggc gaattactta 1020

acgcctcgat tatgttcttt gcgccactga tcattaatcg catcggtggg aaaaacgccc 1080

tgctgctggc tggcactatt atgtctgtac gtattattgg ctcatcgttc gccacctcag 1140

cgctggaagt ggttattctg aaaacgctgc atatgtttga agtaccgttc ctgctggtgg 1200

gctgctttaa atatattacc agccagtttg aagtgcgttt ttcagcgacg atttatctgg 1260

tctgtttctg cttctttaag caactggcga tgatttttat gtctgtactg gcgggcaata 1320

tgtatgaaag catcggtttc cagggcgctt atctggtgct gggtctggtg gcgctgggct 1380

tcaccttaat ttccgtgttc acgcttagcg gccccggccc gctttccctg ctgcgtcgtc 1440

aggtgaatga agtcgcttaa 1460

<210> 2

<211> 4432

<212> DNA

<213> Artificial sequence

<220>

<223> Genetic constructs detecting expressed lactose transporters

<400> 2

atgtactatt taaaaaacac aaacttttgg atgttcggtt tattcttttt cttttacttt 60

tttatcatgg gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc 120

agcaaaagtg atacgggtat tatttttgcc gctatttctc tgttctcgct attattccaa 180

ccgctgtttg gtctgctttc tgacaaactc gggctgcgca aatacctgct gtggattatt 240

accggcatgt tagtgatgtt tgcgccgttc tttattttta tcttcgggcc actgttacaa 300

tacaacattt tagtaggatc gattgttggt ggtatttatc taggcttttg ttttaacgcc 360

ggtgcgccag cagtagaggc atttattgag aaagtcagcc gtcgcagtaa tttcgaattt 420

ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc 480

atgttcacca tcaataatca gtttgttttc tggctgggct ctggctgtgc actcatcctc 540

gccgttttac tctttttcgc caaaacggat gcgccctctt ctgccacggt tgccaatgcg 600

gtaggtgcca accattcggc atttagcctt aagctggcac tggaactgtt cagacagcca 660

aaactgtggt ttttgtcact gtatgttatt ggcgtttcct gcacctacga tgtttttgac 720

caacagtttg ctaatttctt tacttcgttc tttgctaccg gtgaacaggg tacgcgggta 780

tttggctacg taacgacaat gggcgaatta cttaacgcct cgattatgtt ctttgcgcca 840

ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc tggctggcac tattatgtct 900

gtacgtatta ttggctcatc gttcgccacc tcagcgctgg aagtggttat tctgaaaacg 960

ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct ttaaatatat taccagccag 1020

tttgaagtgc gtttttcagc gacgatttat ctggtctgtt tctgcttctt taagcaactg 1080

gcgatgattt ttatgtctgt actggcgggc aatatgtatg aaagcatcgg tttccagggc 1140

gcttatctgg tgctgggtct ggtggcgctg ggcttcacct taatttccgt gttcacgctt 1200

agcggccccg gcccgctttc cctgctgcgt cgtcaggtga atgaagtcgc tgataaaaac 1260

tggaggcgtc ataggggatc ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 1320

tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 1380

ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc 1440

ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc ttcctgaggc 1500

cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc ccatctacac 1560

caacgtgacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga atccgacggg 1620

ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc agacgcgaat 1680

tatttttgat ggcgttaact cggcgtttca tctgtggtgc aacgggcgct gggtcggtta 1740

cggccaggac agtcgtttgc cgtctgaatt tgacctgagc gcatttttac gcgccggaga 1800

aaaccgcctc gcggtgatgg tgctgcgctg gagtgacggc agttatctgg aagatcagga 1860

tatgtggcgg atgagcggca ttttccgtga cgtctcgttg ctgcataaac cgactacaca 1920

aatcagcgat ttccatgttg ccactcgctt taatgatgat ttcagccgcg ctgtactgga 1980

ggctgaagtt cagatgtgcg gcgagttgcg tgactaccta cgggtaacag tttctttatg 2040

gcagggtgaa acgcaggtcg ccagcggcac cgcgcctttc ggcggtgaaa ttatcgatga 2100

gcgtggtggt tatgccgatc gcgtcacact acgtctgaac gtcgaaaacc cgaaactgtg 2160

gagcgccgaa atcccgaatc tctatcgtgc ggtggttgaa ctgcacaccg ccgacggcac 2220

gctgattgaa gcagaagcct gcgatgtcgg tttccgcgag gtgcggattg aaaatggtct 2280

gctgctgctg aacggcaagc cgttgctgat tcgaggcgtt aaccgtcacg agcatcatcc 2340

tctgcatggt caggtcatgg atgagcagac gatggtgcag gatatcctgc tgatgaagca 2400

gaacaacttt aacgccgtgc gctgttcgca ttatccgaac catccgctgt ggtacacgct 2460

gtgcgaccgc tacggcctgt atgtggtgga tgaagccaat attgaaaccc acggcatggt 2520

gccaatgaat cgtctgaccg atgatccgcg ctggctaccg gcgatgagcg aacgcgtaac 2580

gcgaatggtg cagcgcgatc gtaatcaccc gagtgtgatc atctggtcgc tggggaatga 2640

atcaggccac ggcgctaatc acgacgcgct gtatcgctgg atcaaatctg tcgatccttc 2700

ccgcccggtg cagtatgaag gcggcggagc cgacaccacg gccaccgata ttatttgccc 2760

gatgtacgcg cgcgtggatg aagaccagcc cttcccggct gtgccgaaat ggtccatcaa 2820

aaaatggctt tcgctacctg gagagacgcg cccgctgatc ctttgcgaat acgcccacgc 2880

gatgggtaac agtcttggcg gtttcgctaa atactggcag gcgtttcgtc agtatccccg 2940

tttacagggc ggcttcgtct gggactgggt ggatcagtcg ctgattaaat atgatgaaaa 3000

cggcaacccg tggtcggctt acggcggtga ttttggcgat acgccgaacg atcgccagtt 3060

ctgtatgaac ggtctggtct ttgccgaccg cacgccgcat ccagcgctga cggaagcaaa 3120

acaccagcag cagtttttcc agttccgttt atccgggcaa accatcgaag tgaccagcga 3180

atacctgttc cgtcatagcg ataacgagct cctgcactgg atggtggcgc tggatggtaa 3240

gccgctggca agcggtgaag tgcctctgga tgtcgctcca caaggtaaac agttgattga 3300

actgcctgaa ctaccgcagc cggagagcgc cgggcaactc tggctcacag tacgcgtagt 3360

gcaaccgaac gcgaccgcat ggtcagaagc cgggcacatc agcgcctggc agcagtggcg 3420

tctggcggaa aacctcagtg tgacgctccc cgccgcgtcc cacgccatcc cgcatctgac 3480

caccagcgaa atggattttt gcatcgagct gggtaataag cgttggcaat ttaaccgcca 3540

gtcaggcttt ctttcacaga tgtggattgg cgataaaaaa caactgctga cgccgctgcg 3600

cgatcagttc acccgtgcac cgctggataa cgacattggc gtaagtgaag cgacccgcat 3660

tgaccctaac gcctgggtcg aacgctggaa ggcggcgggc cattaccagg ccgaagcagc 3720

gttgttgcag tgcacggcag atacacttgc tgatgcggtg ctgattacga ccgctcacgc 3780

gtggcagcat caggggaaaa ccttatttat cagccggaaa acctaccgga ttgatggtag 3840

tggtcaaatg gcgattaccg ttgatgttga agtggcgagc gatacaccgc atccggcgcg 3900

gattggcctg aactgccagc tggcgcaggt agcagagcgg gtaaactggc tcggattagg 3960

gccgcaagaa aactatcccg accgccttac tgccgcctgt tttgaccgct gggatctgcc 4020

attgtcagac atgtataccc cgtacgtctt cccgagcgaa aacggtctgc gctgcgggac 4080

gcgcgaattg aattatggcc cacaccagtg gcgcggcgac ttccagttca acatcagccg 4140

ctacagtcaa cagcaactga tggaaaccag ccatcgccat ctgctgcacg cggaagaagg 4200

cacatggctg aatatcgacg gtttccatat ggggattggt ggcgacgact cctggagccc 4260

gtcagtatcg gcggaattcc agctgagcgc cggtcgctac cattaccagt tggtctggtg 4320

tcaaaaataa gctggtttga agggtattgg tcggtcagtt tcacctgatt tacgtaaaaa 4380

cccgcttcgg cgggtttttg cttttggagg ggcagaaaga tgaatgactg tc 4432

<210> 3

<211> 2093

<212> DNA

<213> Artificial sequence

<220>

<223> Genetic constructs detecting expressed lactose transporters

<400> 3

atgtactatt taaaaaacac aaacttttgg atgttcggtt tattcttttt cttttacttt 60

tttatcatgg gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc 120

agcaaaagtg atacgggtat tatttttgcc gctatttctc tgttctcgct attattccaa 180

ccgctgtttg gtctgctttc tgacaaactc gggctgcgca aatacctgct gtggattatt 240

accggcatgt tagtgatgtt tgcgccgttc tttattttta tcttcgggcc actgttacaa 300

tacaacattt tagtaggatc gattgttggt ggtatttatc taggcttttg ttttaacgcc 360

ggtgcgccag cagtagaggc atttattgag aaagtcagcc gtcgcagtaa tttcgaattt 420

ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc 480

atgttcacca tcaataatca gtttgttttc tggctgggct ctggctgtgc actcatcctc 540

gccgttttac tctttttcgc caaaacggat gcgccctctt ctgccacggt tgccaatgcg 600

gtaggtgcca accattcggc atttagcctt aagctggcac tggaactgtt cagacagcca 660

aaactgtggt ttttgtcact gtatgttatt ggcgtttcct gcacctacga tgtttttgac 720

caacagtttg ctaatttctt tacttcgttc tttgctaccg gtgaacaggg tacgcgggta 780

tttggctacg taacgacaat gggcgaatta cttaacgcct cgattatgtt ctttgcgcca 840

ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc tggctggcac tattatgtct 900

gtacgtatta ttggctcatc gttcgccacc tcagcgctgg aagtggttat tctgaaaacg 960

ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct ttaaatatat taccagccag 1020

tttgaagtgc gtttttcagc gacgatttat ctggtctgtt tctgcttctt taagcaactg 1080

gcgatgattt ttatgtctgt actggcgggc aatatgtatg aaagcatcgg tttccagggc 1140

gcttatctgg tgctgggtct ggtggcgctg ggcttcacct taatttccgt gttcacgctt 1200

agcggccccg gcccgctttc cctgctgcgt cgtcaggtga atgaagtcgc tcatcatcac 1260

caccatcatt aggatggtgg tgatgataat ggagaaaaaa atcactggat ataccaccgt 1320

tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg 1380

tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa 1440

taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc 1500

ggagttccgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg 1560

ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga 1620

cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct 1680

ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtcagcgcca atccctgggt 1740

gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt 1800

cactatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt 1860

tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta 1920

ctgcgatgag tggcagggcg gggcgtaagg atccaaaggt acctctagag tcgacctgca 1980

ggccttcgta aatctggcga gtggggaact gccagacatc aaataaaaca aaaggctcag 2040

tcggaagact gggccttttg ttttatctgt tgtttgtcgg tgaacactct ccc 2093

<210> 4

<211> 2628

<212> DNA

<213> Artificial sequence

<220>

<223> Genetic constructs detecting expressed lactose transporters

<400> 4

atggcagatc attcgagcag ctcatcttcg ctgcagaaga agccaattaa tactatcgag 60

cataaagaca ctttgggcaa tgatcgggat cacaaggaag ccttgaacag tgataatgat 120

aatacttctg gattgaaaat caatggtgtc cccatcgagg acgctagaga ggaagtgctc 180

ttaccaggtt acttgtcgaa gcaatattac aaattgtacg gtttatgttt tataacatat 240

ctgtgtgcta ctatgcaagg ttatgatggg gctttaatgg gttctatcta taccgaagat 300

gcatatttga aatactacca tttggatatt aactcatcct ctggtactgg tctagtgttc 360

tctattttca acgttggtca aatttgcggt gcattctttg ttcctcttat ggattggaaa 420

ggtagaaaac ctgctatttt aattgggtgt ctgggtgttg ttattggtgc tattatttcg 480

tctttaacaa caacaaagag tgcattaatt ggtggtagat ggttcgtggc ctttttcgct 540

acaatcgcta atgcagcagc tccaacatac tgtgcagaag tggctccagc tcacttaaga 600

ggtaaggttg caggtcttta taacaccctt tggtctgtcg gttccattgt tgctgccttt 660

agcacttacg gtaccaacaa aaacttccct aactcctcca aggcttttaa gattccatta 720

tacttacaaa tgatgttccc aggtcttgtg tgtatatttg gttggttaat cccagaatct 780

ccaagatggt tggttggtgt tggccgtgag gaagaagctc gtgaattcat tatcaaatac 840

cacttaaatg gcgatagaac tcatccatta ttggatatgg agatggcaga aataatagaa 900

tctttccatg gtacagattt atcaaaccct ctagaaatgt tagatgtaag gagcttattc 960

agaacgagat cggataggta cagagcaatg ttggttatac ttatggcttg gttcggtcaa 1020

ttttccggta acaatgtgtg ttcgtactat ttgcctacca tgttgagaaa tgttggtatg 1080

aagagtgtct cattgaatgt gttaatgaat ggtgtttatt ccatcgtcac ttggatttct 1140

tcaatttgcg gtgcattctt tattgataag attggtagaa gggaaggttt ccttggttct 1200

atctcaggtg ctgcattagc attgacaggt ctatctatct gtactgctcg ttatgagaag 1260

actaagaaga agagtgcttc caatggtgca ttggtgttca tttatctctt tggtggtatc 1320

ttttcttttg ctttcactcc aatgcaatcc atgtactcaa cagaagtgtc tacaaacttg 1380

acgagatcta aggcccaact cctcaacttt gtggtttctg gtgttgccca atttgttaat 1440

caatttgcta ctccaaaggc aatgaagaat atcaaatatt ggttctatgt gttctacgtt 1500

ttcttcgata ttttcgaatt tattgttatc tacttcttct tcgttgaaac taagggtaga 1560

agcttagaag aattagaagt tgtctttgaa gctccaaacc caagaaaggc atccgttgat 1620

caagcattct tggctcaagt cagggcaact ttggtccaac gaaatgacgt tagagttgca 1680

aatgctcaaa atttgaaaga gcaagagcct ctaaagagcg atgctgatca tgtcgaaaag 1740

ctttcagagg cagaatctgt tagagcagaa ggaaggggtt ctttgttgac ttgtggagat 1800

gttgaggaga atccaggacc aggtaaggaa aagactcacg tttcgaggcc gcgattaaat 1860

tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca 1920

ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat 1980

ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg 2040

gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta 2100

ctcaccactg cgatccccgg caaaacagca ttccaggtat tagaagaata tcctgattca 2160

ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt 2220

tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg 2280

aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa 2340

caagtctgga aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat 2400

ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat 2460

gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc 2520

ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct 2580

gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaa 2628

<210> 5

<211> 1457

<212> DNA

<213> Artificial sequence

<220>

<223> Genetic constructs detecting expressed lactose transporters

<400> 5

atgtactatt taaaaaacac aaacttttgg atgttcggtt tattcttttt cttttacttt 60

tttatcatgg gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc 120

agcaaaagtg atacgggtat tatttttgcc gctatttctc tgttctcgct attattccaa 180

ccgctgtttg gtctgctttc tgacaaactc gggctgcgca aatacctgct gtggattatt 240

accggcatgt tagtgatgtt tgcgccgttc tttattttta tcttcgggcc actgttacaa 300

tacaacattt tagtaggatc gattgttggt ggtatttatc taggcttttg ttttaacgcc 360

ggtgcgccag cagtagaggc atttattgag aaagtcagcc gtcgcagtaa tttcgaattt 420

ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc 480

atgttcacca tcaataatca gtttgttttc tggctgggct ctggctgtgc actcatcctc 540

gccgttttac tctttttcgc caaaacggat gcgccctctt ctgccacggt tgccaatgcg 600

gtaggtgcca accattcggc atttagcctt aagctggcac tggaactgtt cagacagcca 660

aaactgtggt ttttgtcact gtatgttatt ggcgtttcct gcacctacga tgtttttgac 720

caacagtttg ctaatttctt tacttcgttc tttgctaccg gtgaacaggg tacgcgggta 780

tttggctacg taacgacaat gggcgaatta cttaacgcct cgattatgtt ctttgcgcca 840

ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc tggctggcac tattatgtct 900

gtacgtatta ttggctcatc gttcgccacc tcagcgctgg aagtggttat tctgaaaacg 960

ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct ttaaatatat taccagccag 1020

tttgaagtgc gtttttcagc gacgatttat ctggtctgtt tctgcttctt taagcaactg 1080

gcgatgattt ttatgtctgt actggcgggc aatatgtatg aaagcatcgg tttccagggc 1140

gcttatctgg tgctgggtct ggtggcgctg ggcttcacct taatttccgt gttcacgctt 1200

agcggccccg gcccgctttc cctgctgcgt cgtcaggtga atgaagtcgc tgataactct 1260

acgacaacct cttcacagcc aatctcgccc ggatagctca gtcggtagag cagcggccgg 1320

acgcaactga atgaaatggt gaaggacggg tccaggtgtg gctgcttcgg cagtgcagct 1380

tgttgagtag agtgtgagct ccgtaactag tcgcgtccgg ccgcgggtcc agggttcaag 1440

tccctgttcg ggcgcca 1457

<210> 6

<211> 3638

<212> DNA

<213> Artificial sequence

<220>

<223> Vector

<400> 6

cgcgttggat gcaggcatgc aagcttggct gttttggcgg atgagagaag attttcagcc 60

tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca 120

gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg 180

atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga 240

aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 300

ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg 360

tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg 420

acggatggcc tttttgcgtt tctacaaact ctttttgttt atttttctaa atacattcaa 480

atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 540

agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 600

ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 660

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 720

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 780

tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 840

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 900

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 960

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 1020

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 1080

cgatgcctac agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 1140

tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 1200

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 1260

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 1320

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 1380

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1440

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 1500

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1560

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1620

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1680

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 1740

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 1800

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 1860

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 1920

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 1980

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 2040

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 2100

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 2160

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 2220

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 2280

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 2340

cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 2400

tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc 2460

gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc 2520

gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 2580

gagagctcga tatcccgggc ggccgccttc attctataag tttcttgaca tcttggccgg 2640

catatggtat aatagggaaa tttccatggc ggccgctcta gaagaagctt gggatccgtc 2700

gacctcgaat tcggaggaaa caaagatggc ctttaaagtt gttcagattt gtggtggtct 2760

gggcaatcag atgtttcagt atgcatttgc aaaaagcctg cagaaacata gcaatacacc 2820

ggttctgctg gatattacca gctttgattg gagcaatcgt aaaatgcagc tggaactgtt 2880

tccgattgat ctgccgtatg caagcgaaaa agaaattgca attgccaaaa tgcagcatct 2940

gccgaaactg gttcgtaatg ttctgaaatg catgggtttt gatcgtgtga gccaagaaat 3000

cgtgtttgaa tatgaaccga aactgctgaa aaccagccgt ctgacctatt tttatggcta 3060

ttttcaggat ccgcgttatt ttgatgcaat tagtccgctg atcaaacaga cctttaccct 3120

gcctccgcct ccggaaaatg gtaataacaa aaaaaaagaa gaagagtatc atcgtaaact 3180

ggcactgatt ctggcagcaa aaaatagcgt gtttgtgcat attcgtcgcg gtgattatgt 3240

tggtattggt tgtcagctgg gcatcgatta tcagaaaaaa gcactggaat acatggcaaa 3300

acgtgttccg aatatggaac tgtttgtgtt ttgcgaggac ctggaattta cccagaatct 3360

ggatctgggc tatccgttta tggatatgac cacccgtgat aaagaggaag aggcatattg 3420

ggatatgctg ctgatgcaga gctgtaaaca tggtattatt gccaacagca cctatagttg 3480

gtgggcagca tatctgatta ataacccgga aaaaatcatt attggtccga aacattggct 3540

gtttggccat gaaaacatcc tgtgtaaaga atgggtgaaa atcgaaagcc actttgaagt 3600

gaaaagccag aaatataatg cctaataaga gctcccaa 3638

<210> 7

<211> 2660

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 7

tattttagat tcctgacttc aactcaagac gcacagatat tataacatct gcagaatagg 60

catttgcaag aattactcgt gagtaaggaa agagtgagga actatcgcat acctgcattt 120

aaagatgccg atttgggcgc gaatccttta ttttggcttc accctcatac tattatcagg 180

gccagaaaaa ggaagtgttt ccctccttct tgaattgatg ttaccctcat aaagcacgtg 240

gcctcttatc gagaaagaaa ttaccgtcgc tcgtgatttg tttgcaaaaa gaacaaaact 300

gaaaaaaccc agacacgctc gacttcctct cttcctattg attgcagctt ccaatttcgt 360

cacacaacaa ggtcctagcg acggctcaca ggttttgtaa caagcaatcg aaggttctgg 420

aatggcggga aagggtttag taccacatgc tatgatgccc actgtgatca ccagagcaaa 480

gttcgttcga tcgtactgtt actctctctc tttcaaacag aattgtccga atcgtgtgac 540

aacaacagcc tgttctcaca cactcttttc ttctaaccaa gggggtggtt tagtttagta 600

gaacctcgtg aaacttacat ttacatatat ataaacttgc ataaattggt caatgcaaga 660

aatacatatt tggtcttttc taattcgtag ttttttcaag ttcttagatg ctttcttttt 720

ctcttttttt acagatcatc aaggaagtaa ttatctactt tttacaacaa atataaaaaa 780

aatggcagat cattcgagca gctcatcttc gctgcagaag aagccaatta atactatcga 840

gcataaagac actttgggca ttgatctgga tcacaaggaa gccttgaaca gtgataatga 900

taatacttct ggattgaaaa tcaatggtgt ccccatcgag gacgctagag aggaagtgct 960

cttaccaggt tacttgtcga agcaatatta caaattgtac agtttatgtt ttgtaacata 1020

tctgtgtgct actatgcaag gttatgatgg ggctttaatg ggttctatct ataccgaaaa 1080

tgcatatttg gaatactacc atttggatat taactcatcc agtggtactg gtctagtgtt 1140

ctctattttc aacgttggtc aaatttgcgg tgcattcttt gttcctctta tggattggaa 1200

aggtagaaaa cctgctattt taattgggtg tctgggtgtt gttattggtg gtattattac 1260

gtctgtaaca acaacaaaga gtgcattaat tggtggtaga tggttcatgg cctttttcgc 1320

tacaatcgct aattcagcag ctccagcata ctgtgcagaa gtggctccag ctcacttaag 1380

aggtaaggtt gcaggtcttt ataacaccct ttggtctgtc ggttccattg ttgctgcctt 1440

taccactctc ggtaccaaca aaaacttccc taactcctcc aaggctttta agattccatt 1500

atacttacaa atgatgttcc caggtcttgt gtgtatattt ggttggttaa tcccagaatc 1560

tccaagatgg ttggttggtg ttggccgtga ggaagaagct cgtgaattca ttatcaaata 1620

ccacttaaat ggcgatagaa ctcatccatt attggatatg gagatggcag aaataataga 1680

atctttccat ggtacagatt tatcaaaccc tctagaaatg ttagatgtaa ggatcttatt 1740

cagaacgaga tcggataggt acagagcaat gttggttata cttatggctt ggttcggtca 1800

attttccggt aacaatgtgt gttcgtacta tttgcctacc atgttgagaa atgttggtat 1860

gaagagtgtc tcattgaatg tgttaatgaa tggtgtttat tccatcgtct cttggatttc 1920

ttcaatttgc ggtgcattct ttattgataa gattggtaga agggaaggtt tccttggttc 1980

tatctcaggt gctgcattag cattgacagg tctatctatc tgtactgctc gttatgagaa 2040

gactaagaag aagagtgctt ccaatggtgc attggtgttc atttatctct ttggtgttat 2100

cttttctttt gctttcactc caatgcaatc catgtactca acagaagtgt ctacaaactt 2160

gacgagatct aaggcccaac tcctcaacgg tgtggtttct ggtgttgccc aatttgttaa 2220

tcaatttgct actccaaagg caatgaagaa tatcaaatat tggttctatg tgttctacgt 2280

tttcttcgat attttcgaat ttattgttat ctacttcttc ttcgttgaaa ctaagggtag 2340

aagcttagaa gaattagaag ctgtctttga agctccaaac ccaagaaagg catccgttga 2400

tcaagcattc ttggctcaag ccagggcaac tttggtccaa caaaatgacg ttagagttgc 2460

aaatgctcaa aatttgaaag agcaagagct tctaaagagc gatgctgatc atgtcgaaaa 2520

gctttcagag gcagaatctg tttaaagagt cttttgtaac gaccccgtct ccaccaactt 2580

ggtatgcttg aaatctcaag gccattacac attcagttat gtgaacgaaa ggtctttatt 2640

taacgtagca taaactaaat 2660

<210> 8

<211> 2554

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 8

aggaataagg atacttcaag actagattcc cccctgcatt cccatcagaa ccgtaaacct 60

tggcgctttc cttgggaagt attcaagaag tgccttgtcc ggtttctgtg gctcacaaac 120

cagcgcgccc gatatggctt tcttttcact tatgaatgta ccagtacggg acaattagaa 180

cgctcctgta acaatctctt tgcaaatgtg gggttacatt ctaaccatgt cacactgctg 240

acgaaattca aagtaaaaaa aaatgggacc acgtcttgag aacgatagat tttctttatt 300

ttacattgaa cagtcgttgt ctcagcgcgc tttatgtttt cattcatact tcatattata 360

aaataacaaa agaagaattt catattcacg cccaagaaat caggctgctt tccaaatgca 420

attgacactt cattagccat cacacaaaac tctttcttgc tggagcttct tttaaaaaag 480

acctcagtac accaaacacg ttacccgacc tcgttatttt acgacaacta tgataaaatt 540

ctgaagaaaa aataaaaaaa ttttcatact tcttgctttt atttaaacca ttgaatgatt 600

tcttttgaac aaaactacct gtttcaccaa aggaaataga aagaaaaaat caattagaag 660

aaaacaaaaa acaaaatggc agatcattcg agcagctcat cttcgctgca gaagaagcca 720

attaatacta tcgagcataa agacactttg ggcattgatc tggatcacaa ggaagccttg 780

aacagtgata atgataatac ttctggattg aaaatcaatg gtgtccccat cgaggacgct 840

agagaggaag tgctcttacc aggttacttg tcgaagcaat attacaaatt gtacagttta 900

tgttttgtaa catatctgtg tgctactatg caaggttatg atggggcttt aatgggttct 960

atctataccg aaaatgcata tttggaatac taccatttgg atattaactc atccagtggt 1020

actggtctag tgttctctat tttcaacgtt ggtcaaattt gcggtgcatt ctttgttcct 1080

cttatggatt ggaaaggtag aaaacctgct attttaattg ggtgtctggg tgttgttatt 1140

ggtggtatta ttacgtctgt aacaacaaca aagagtgcat taattggtgg tagatggttc 1200

atggcctttt tcgctacaat cgctaattca gcagctccag catactgtgc agaagtggct 1260

ccagctcact taagaggtaa ggttgcaggt ctttataaca ccctttggtc tgtcggttcc 1320

attgttgctg cctttaccac tctcggtacc aacaaaaact tccctaactc ctccaaggct 1380

tttaagattc cattatactt acaaatgatg ttcccaggtc ttgtgtgtat atttggttgg 1440

ttaatcccag aatctccaag atggttggtt ggtgttggcc gtgaggaaga agctcgtgaa 1500

ttcattatca aataccactt aaatggcgat agaactcatc cattattgga tatggagatg 1560

gcagaaataa tagaatcttt ccatggtaca gatttatcaa accctctaga aatgttagat 1620

gtaaggatct tattcagaac gagatcggat aggtacagag caatgttggt tatacttatg 1680

gcttggttcg gtcaattttc cggtaacaat gtgtgttcgt actatttgcc taccatgttg 1740

agaaatgttg gtatgaagag tgtctcattg aatgtgttaa tgaatggtgt ttattccatc 1800

gtctcttgga tttcttcaat ttgcggtgca ttctttattg ataagattgg tagaagggaa 1860

ggtttccttg gttctatctc aggtgctgca ttagcattga caggtctatc tatctgtact 1920

gctcgttatg agaagactaa gaagaagagt gcttccaatg gtgcattggt gttcatttat 1980

ctctttggtg ttatcttttc ttttgctttc actccaatgc aatccatgta ctcaacagaa 2040

gtgtctacaa acttgacgag atctaaggcc caactcctca acggtgtggt ttctggtgtt 2100

gcccaatttg ttaatcaatt tgctactcca aaggcaatga agaatatcaa atattggttc 2160

tatgtgttct acgttttctt cgatattttc gaatttattg ttatctactt cttcttcgtt 2220

gaaactaagg gtagaagctt agaagaatta gaagctgtct ttgaagctcc aaacccaaga 2280

aaggcatccg ttgatcaagc attcttggct caagccaggg caactttggt ccaacaaaat 2340

gacgttagag ttgcaaatgc tcaaaatttg aaagagcaag agcttctaaa gagcgatgct 2400

gatcatgtcg aaaagctttc agaggcagaa tctgtttaaa gagtcttttg taacgacccc 2460

gtctccacca acttggtatg cttgaaatct caaggccatt acacattcag ttatgtgaac 2520

gaaaggtctt tatttaacgt agcataaact aaat 2554

<210> 9

<211> 4141

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 9

tattttagat tcctgacttc aactcaagac gcacagatat tataacatct gcagaatagg 60

catttgcaag aattactcgt gagtaaggaa agagtgagga actatcgcat acctgcattt 120

aaagatgccg atttgggcgc gaatccttta ttttggcttc accctcatac tattatcagg 180

gccagaaaaa ggaagtgttt ccctccttct tgaattgatg ttaccctcat aaagcacgtg 240

gcctcttatc gagaaagaaa ttaccgtcgc tcgtgatttg tttgcaaaaa gaacaaaact 300

gaaaaaaccc agacacgctc gacttcctct cttcctattg attgcagctt ccaatttcgt 360

cacacaacaa ggtcctagcg acggctcaca ggttttgtaa caagcaatcg aaggttctgg 420

aatggcggga aagggtttag taccacatgc tatgatgccc actgtgatca ccagagcaaa 480

gttcgttcga tcgtactgtt actctctctc tttcaaacag aattgtccga atcgtgtgac 540

aacaacagcc tgttctcaca cactcttttc ttctaaccaa gggggtggtt tagtttagta 600

gaacctcgtg aaacttacat ttacatatat ataaacttgc ataaattggt caatgcaaga 660

aatacatatt tggtcttttc taattcgtag ttttttcaag ttcttagatg ctttcttttt 720

ctcttttttt acagatcatc aaggaagtaa ttatctactt tttacaacaa atataaaaaa 780

aatgtcttgc cttattcctg agaatttaag gaaccccaaa aaggttcacg aaaatagatt 840

gcctactagg gcttactact atgatcagga tattttcgaa tctctcaatg ggccttgggc 900

ttttgcgttg tttgatgcac ctcttgacgc tccggatgct aagaatttag actgggaaac 960

ggcaaagaaa tggagcacca tttctgtgcc atcccattgg gaacttcagg aagactggaa 1020

gtacggtaaa ccaatttaca cgaacgtaca gtaccctatc ccaatcgaca tcccaaatcc 1080

tcccactgta aatcctactg gtgtttatgc tagaactttt gaattagatt cgaaatcgat 1140

tgagtcgttc gagcacagat tgagatttga gggtgtggac aattgttacg agctttatgt 1200

taatggtcaa tatgtgggtt tcaataaggg gtcccgtaac ggggctgaat ttgatatcca 1260

aaagtacgtt tctgagggcg aaaacttagt ggtcgtcaag gttttcaagt ggtccgattc 1320

cacttatatc gaggaccaag atcaatggtg gctctctggt atttacagag acgtttcttt 1380

actaaaattg cctaagaagg cccatattga agacgttagg gtcactacaa cttttgtgga 1440

ctctcagtat caggatgcag agctttctgt gaaagttgat gtccagggtt cttcttatga 1500

tcacatcaat ttcacacttt acgaacctga agatggatct aaagtttacg atgcaagctc 1560

tttgttgaac gaggagaatg ggaacacgac tttttcaact aaagaattta tttccttctc 1620

caccaaaaag aacgaagaaa cagctttcaa gatcaacgtc aaggccccag aacattggac 1680

cgcagaaaat cctactttgt acaagtacca gttggattta attggatctg atggcagtgt 1740

gattcaatct attaagcacc atgttggttt cagacaagtg gagttgaagg acggtaacat 1800

tactgttaat ggcaaagaca ttctctttag aggtgtcaac agacatgatc accatccaag 1860

gttcggtaga gctgtgccat tagattttgt tgttagggac ttgattctaa tgaagaagtt 1920

taacatcaat gctgttcgta actcgcatta tccaaaccat cctaaggtgt atgacctctt 1980

cgataagctg ggcttctggg tcattgacga ggcagatctt gaaactcatg gtgttcaaga 2040

gccatttaat cgtcatacga acttggaggc tgaatatcca gatactaaaa ataaactcta 2100

cgatgttaat gcccattact tatcagataa tccagagtac gaggtcgcgt acttagacag 2160

agcttcccaa cttgtcctaa gagatgtcaa tcatccttcg attattatct ggtccttggg 2220

taacgaagct tgttatggca gaaaccacaa agccatgtac aagttaatta aacaattgga 2280

tcctaccaga cttgtgcatt atgagggtga cttgaacgct ttgagtgcag atatctttag 2340

tttcatgtac ccaacatttg aaattatgga aaggtggagg aagaaccaca ctgatgaaaa 2400

tggtaagttt gaaaagcctt tgatcttgtg tgagtacggc catgcaatgg gtaacggtcc 2460

tggctctttg aaagaatatc aagagttgtt ctacaaggag aagttttacc aaggtggctt 2520

tatctgggaa tgggcaaatc acggtattga attcgaagat gttagtactg cagatggtaa 2580

gttgcataaa gcttatgctt atggtggtga ctttaaggaa gaggttcatg acggagtgtt 2640

catcatggat ggtttgtgta acagtgagca taatcctact ccgggccttg tagagtataa 2700

gaaggttatt gaacccgttc atattaaaat tgcgcacgga tctgtaacaa tcacaaataa 2760

gcacgacttc attacgacag accacttatt gtttatcgac aaggacacgg gaaagacaat 2820

cgacgttcca tctttaaagc cagaagaatc tgttactatt ccttctgata caacttatgt 2880

tgttgccgtg ttgaaagatg atgctggtgt tctaaaggca ggtcatgaaa ttgcctgggg 2940

ccaagctgaa cttccattga aggtacccga ttttgttaca gagacagcag aaaaagctgc 3000

gaagatcaac gacggtaaac gttatgtctc agttgaatcc agtggattgc attttatctt 3060

ggacaaattg ttgggtaaaa ttgaaagcct aaaggtcaag ggtaaggaaa tttccagcaa 3120

gtttgagggt tcttcaatca ctttctggag acctccaacg aataatgatg aacctaggga 3180

ctttaagaac tggaagaagt acaatattga tttaatgaag caaaacatcc atggagtgag 3240

tgtcgaaaaa ggttctaatg gttctctagc tgtagtcacg gttaactctc gtatatcccc 3300

agttgtattt tactatgggt ttgagactgt tcagaagtac acgatctttg ctaacaaaat 3360

aaacttgaac acttctatga agcttactgg cgaatatcag cctcctgatt tcccaagagt 3420

tgggtacgaa ttctggctag gagatagtta tgaatcattt gaatggttag gtcgcgggcc 3480

cggcgaatca tatccggata agaaggaatc tcaaagattc ggtctttacg attccaaaga 3540

tgtagaggaa ttcgtatatg actatcctca agaaaatgga aatcatacag atacccactt 3600

tttgaacatc aaatttgaag gtgcaggaaa actatcgatc ttccaaaagg agaagccatt 3660

taacttcaag atttcagacg aatacggggt tgatgaagct gcccacgctt gtgacgttaa 3720

aagatacggc agacactatc taaggttgga ccatgcaatc catggtgttg gtagcgaagc 3780

atgcggacct gctgttctgg accagtacag attgaaagct caagatttca actttgagtt 3840

tgatctcgct tttgaataag ccggccattg aattgaattg aaatcgatag atcaattttt 3900

ttcttttctc tttccccatc ctttacgcta aaataatagt ttattttatt ttttgaatat 3960

tttttattta tatacgtata tatagactat tatttatctt ttaatgatta ttaagatttt 4020

tattaaaaaa aaattcgcac ctcttttaat gcctttatgc agtttttttt tcccattcga 4080

tatttctatg ttcgggttca gcgtatttta agtttaataa ctcgaaaatt ctgcgtttcg 4140

a 4141

<210> 10

<211> 1187

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 10

ttaatctcag cagatcgtaa caacaaggct actctactgc ttacaatacc ccgttgtaca 60

tctaagtcgt atacaaatga tttatcccca cgcaaaatga cattgcaatt cgccagcaag 120

cacccaaggc ctttccgcca agtgcaccgt tgctagcctg ctatggttca gcgacgccac 180

aaggacgcct tattcgtatc catctatatt gtgtggagca aagaaatcac cgcgttctag 240

catggattct gacttagagg cgttcagcca taatccagcg gatggtagct tcgcggcaat 300

gcctgatcag acagccgcaa aaaccaatta tccgaatgaa ctgttcctct cgtactaagt 360

tcaattacta ttgcggtaac attcatcagt agggtaaaac taacctgtct cacgacggtc 420

taaacccagc tcacgttccc tattagtggg tgaacaatcc aacgcttacc gaattctgct 480

tcggtatgat aggaagagcc gacatcgaag aatcaaaaag caatgtcgct atgaacgctt 540

gactgccaca agccagttat ccctgtggta acttttctgg cacctctagc ctcaaattcc 600

gagggactaa aggatcgata ggccacactt tcatggtttg tattcacact gaaaatcaaa 660

atcaaggggg cttttaccct tttgttctac tggagatttc tgttctccat gagcccccct 720

taggacatct gcgttatcgt ttaacagatg tgccgcccca gccaaactcc ccacctgaca 780

atgtcttcaa cccggatcag ccccgaatgg gaccttgaat gctagaacgt ggaaaatgaa 840

ttccagctcc gcttcattga ataagtaaag aaactataaa ggtagtggta tttcactggc 900

gccgaagctc ccacttattc tacaccctct atgtctcttc acaatgtcaa actagagtca 960

agctcaacag ggtcttcttt ccccgctgat tctgccaagc ccgttccctt ggctgtggtt 1020

tcgctagata gtagataggg acagtgggaa tctcgttaat ccattcatgc gcgtcactaa 1080

ttagatgacg aggcatttgg ctaccttaag agagtcatag ttactcccgc cgtttacccg 1140

cgcttggttg aatttcttca ctttgacatt cagagcactg ggcagaa 1187

<210> 11

<211> 2010

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 11

agtagtccgc ctagcagagc aagccccacc aagcagtcca caagcacgcc cgctgcgtct 60

gaccaaggcc ctcactaccc gacccttaga gccaatcctt atcccgaagt tacggatcta 120

ttttgccgac ttcccttatc tacattattc tatcaactag aggctgttca ccttggagac 180

ctgctgcggt tatcagtacg acctggcatg aaaactattc cttcctgtgg attttcacgg 240

gccgtcacaa gcgcaccgga gccagcaaag gtgctggcct cttccagcca taagacccca 300

tctccggata aaccaattcc ggggtgataa gctgttaaga agaaaagata actcctccca 360

gggctcgcgc cgacgtctcc acattcagtt acgttaccgt gaagaatcca tatccaggtt 420

ccggaatctt aaccggattc cctttcgatg gtggcctgca taaaatcagg cctttgaaac 480

ggagcttccc catctcttag gatcgactaa cccacgtcca actgctgttg acgtggaacc 540

tttccccact tcagtcttca aagttctcat ttgaatattt gctactacca ccaagatctg 600

cactagaggc cgttcgaccc gaccttacgg tctaggcttc gtcactgacc tccacgcctg 660

cctactcgtc agggcatcat atcaaccctg acggtagagt ataggtaaca cgcttgagcg 720

ccatccattt tcagggctag ttcattcggc cggtgagttg ttacacactc cttagcggat 780

tccgacttcc atggccaccg tccggctgtc tagatgaact aacacctttt gtggtgtctg 840

atgagcgtgt attccggcac cttaactcta cgttcggttc atcccgcatc gccagttctg 900

cttaccaaaa atggcccact aaaagctctt cattcaaatg tccacgttca attaagtaac 960

aaggacttct tacatattta aagtttgaga ataggtcaag gtcatttcga ccccggaacc 1020

tctaatcatt cgctttacct cataaaactg atacgagctt ctgctatcct gagggaaact 1080

tcggcaggaa ccagctacta gatggttcga ttagtctttc gcccctatac ccaaattcga 1140

cgatcgattt gcacgtcaga accgctacga gcctccacca gagtttcctc tggcttcacc 1200

ctattcaggc atagttcacc atctttcggg tcccaacagc tatgctctta ctcaaatcca 1260

tccgaagaca tcaggatcgg tcgattgtgc acctcttgcg aggccccaac ctacgttcac 1320

tttcattacg cgtatgggtt ttacacccaa acactcgcat agacgttaga ctccttggtc 1380

cgtgtttcaa gacgggcggc atataaccat tatgccagca tccttgactt acgtcgcagt 1440

cctcagtccc agctggcagt attcccacag gctataatac ttaccgaggc aagctacatt 1500

cctatggatt tatcctgcca ccaaaactga tgctggccca gtgaaatgcg agattcccct 1560

acccacaagg agcagagggc acaaaacacc atgtctgatc aaatgccctt ccctttcaac 1620

aatttcacgt actttttcac tctcttttca aagttctttt catctttcca tcactgtact 1680

tgttcgctat cggtctctcg ccaatattta gctttagatg gaatttacca cccacttaga 1740

gctgcattcc caaacaactc gactcttcga aggcacttta caaagaaccg cactcctcgc 1800

cacacgggat tctcaccctc tatgacgtcc tgttccaagg aacatagaca aggaacggcc 1860

ccaaagttgc cctctccaaa ttacaactcg ggcaccgaag gtaccagatt tcaaatttga 1920

gcttttgccg cttcactcgc cgttactaag gcaatcccgg ttggtttctt ttcctccgct 1980

tattgatatg cttaagttca gcgggtactc 2010

<210> 12

<211> 1558

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 12

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcttactgt cgggaattcg cgttggccaa ttcattaatg gtataaggtt 180

cttgacatct tacaatcaat atggtataat aatttagtta gggcccaagt tcacttaaaa 240

aggagatcaa caatgaaagc aattttcgta ctgaaacatc ttaatcatgc gtaggatttt 300

ttctatgtac tatttaaaaa acacaaactt ttggatgttc ggtttattct ttttctttta 360

cttttttatc atgggagcct acttcccgtt tttcccgatt tggctacatg acatcaacca 420

tatcagcaaa agtgatacgg gtattatttt tgccgctatt tctctgttct cgctattatt 480

ccaaccgctg tttggtctgc tttctgacaa actcgggctg cgcaaatacc tgctgtggat 540

tattaccggc atgttagtga tgtttgcgcc gttctttatt tttatcttcg ggccactgtt 600

acaatacaac attttagtag gatcgattgt tggtggtatt tatctaggct tttgttttaa 660

cgccggtgcg ccagcagtag aggcatttat tgagaaagtc agccgtcgca gtaatttcga 720

atttggtcgc gcgcggatgt ttggctgtgt tggctgggcg ctgtgtgcct cgattgtcgg 780

catcatgttc accatcaata atcagtttgt tttctggctg ggctctggct gtgcactcat 840

cctcgccgtt ttactctttt tcgccaaaac ggatgcgccc tcttctgcca cggttgccaa 900

tgcggtaggt gccaaccatt cggcatttag ccttaagctg gcactggaac tgttcagaca 960

gccaaaactg tggtttttgt cactgtatgt tattggcgtt tcctgcacct acgatgtttt 1020

tgaccaacag tttgctaatt tctttacttc gttctttgct accggtgaac agggtacgcg 1080

ggtatttggc tacgtaacga caatgggcga attacttaac gcctcgatta tgttctttgc 1140

gccactgatc attaatcgca tcggtgggaa aaacgccctg ctgctggctg gcactattat 1200

gtctgtacgt attattggct catcgttcgc cacctcagcg ctggaagtgg ttattctgaa 1260

aacgctgcat atgtttgaag taccgttcct gctggtgggc tgctttaaat atattaccag 1320

ccagtttgaa gtgcgttttt cagcgacgat ttatctggtc tgtttctgct tctttaagca 1380

actggcgatg atttttatgt ctgtactggc gggcaatatg tatgaaagca tcggtttcca 1440

gggcgcttat ctggtgctgg gtctggtggc gctgggcttc accttaattt ccgtgttcac 1500

gcttagcggc cccggcccgc tttccctgct gcgtcgtcag gtgaatgaag tcgcttaa 1558

<210> 13

<211> 1551

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 13

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacacttac tgtcgggaat tcgcgttggc caattaatta atggtataag gttcttgaca 180

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 240

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 300

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 360

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 420

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 480

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 540

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 600

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 660

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 720

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 780

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 840

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 900

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 960

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 1020

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 1080

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1140

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1200

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1260

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1320

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1380

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1440

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1500

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1551

<210> 14

<211> 1398

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 14

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaattttc gtacatgtac tatttaaaaa acacaaactt ttggatgttc 180

ggtttattct ttttctttta cttttttatc atgggagcct acttcccgtt tttcccgatt 240

tggctacatg acatcaacca tatcagcaaa agtgatacgg gtattatttt tgccgctatt 300

tctctgttct cgctattatt ccaaccgctg tttggtctgc tttctgacaa actcgggctg 360

cgcaaatacc tgctgtggat tattaccggc atgttagtga tgtttgcgcc gttctttatt 420

tttatcttcg ggccactgtt acaatacaac attttagtag gatcgattgt tggtggtatt 480

tatctaggct tttgttttaa cgccggtgcg ccagcagtag aggcatttat tgagaaagtc 540

agccgtcgca gtaatttcga atttggtcgc gcgcggatgt ttggctgtgt tggctgggcg 600

ctgtgtgcct cgattgtcgg catcatgttc accatcaata atcagtttgt tttctggctg 660

ggctctggct gtgcactcat cctcgccgtt ttactctttt tcgccaaaac ggatgcgccc 720

tcttctgcca cggttgccaa tgcggtaggt gccaaccatt cggcatttag ccttaagctg 780

gcactggaac tgttcagaca gccaaaactg tggtttttgt cactgtatgt tattggcgtt 840

tcctgcacct acgatgtttt tgaccaacag tttgctaatt tctttacttc gttctttgct 900

accggtgaac agggtacgcg ggtatttggc tacgtaacga caatgggcga attacttaac 960

gcctcgatta tgttctttgc gccactgatc attaatcgca tcggtgggaa aaacgccctg 1020

ctgctggctg gcactattat gtctgtacgt attattggct catcgttcgc cacctcagcg 1080

ctggaagtgg ttattctgaa aacgctgcat atgtttgaag taccgttcct gctggtgggc 1140

tgctttaaat atattaccag ccagtttgaa gtgcgttttt cagcgacgat ttatctggtc 1200

tgtttctgct tctttaagca actggcgatg atttttatgt ctgtactggc gggcaatatg 1260

tatgaaagca tcggtttcca gggcgcttat ctggtgctgg gtctggtggc gctgggcttc 1320

accttaattt ccgtgttcac gcttagcggc cccggcccgc tttccctgct gcgtcgtcag 1380

gtgaatgaag tcgcttaa 1398

<210> 15

<211> 1558

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 15

cccgtcttac tgtcgggaat tcgcgttggc caattaatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcttactgt cgggaattcg cgttggccga ttcattaatg gtataaggtt 180

cttgacatct tacaatcaat atggtataat aatttagtta gggcccaagt tcacttaaaa 240

aggagatcaa caatgaaagc aattttcgta ctgaaacatc ttaatcatgc gtaggatttt 300

ttctatgtac tatttaaaaa acacaaactt ttggatgttc ggtttattct ttttctttta 360

cttttttatc atgggagcct acttcccgtt tttcccgatt tggctacatg acatcaacca 420

tatcagcaaa agtgatacgg gtattatttt tgccgctatt tctctgttct cgctattatt 480

ccaaccgctg tttggtctgc tttctgacaa actcgggctg cgcaaatacc tgctgtggat 540

tattaccggc atgttagtga tgtttgcgcc gttctttatt tttatcttcg ggccactgtt 600

acaatacaac attttagtag gatcgattgt tggtggtatt tatctaggct tttgttttaa 660

cgccggtgcg ccagcagtag aggcatttat tgagaaagtc agccgtcgca gtaatttcga 720

atttggtcgc gcgcggatgt ttggctgtgt tggctgggcg ctgtgtgcct cgattgtcgg 780

catcatgttc accatcaata atcagtttgt tttctggctg ggctctggct gtgcactcat 840

cctcgccgtt ttactctttt tcgccaaaac ggatgcgccc tcttctgcca cggttgccaa 900

tgcggtaggt gccaaccatt cggcatttag ccttaagctg gcactggaac tgttcagaca 960

gccaaaactg tggtttttgt cactgtatgt tattggcgtt tcctgcacct acgatgtttt 1020

tgaccaacag tttgctaatt tctttacttc gttctttgct accggtgaac agggtacgcg 1080

ggtatttggc tacgtaacga caatgggcga attacttaac gcctcgatta tgttctttgc 1140

gccactgatc attaatcgca tcggtgggaa aaacgccctg ctgctggctg gcactattat 1200

gtctgtacgt attattggct catcgttcgc cacctcagcg ctggaagtgg ttattctgaa 1260

aacgctgcat atgtttgaag taccgttcct gctggtgggc tgctttaaat atattaccag 1320

ccagtttgaa gtgcgttttt cagcgacgat ttatctggtc tgtttctgct tctttaagca 1380

actggcgatg atttttatgt ctgtactggc gggcaatatg tatgaaagca tcggtttcca 1440

gggcgcttat ctggtgctgg gtctggtggc gctgggcttc accttaattt ccgtgttcac 1500

gcttagcggc cccggcccgc tttccctgct gcgtcgtcag gtgaatgaag tcgcttaa 1558

<210> 16

<211> 1558

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 16

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcttactgt cgggaattcg cgttggccga ttcttataaa tcattatctt 180

cttgacattt tagaaacaat atggtataat ataacgataa gggcccaagt tcacttaaaa 240

aggagatcaa caatgaaagc aattttcgta ctgaaacatc ttaatcatgc gtaggatttt 300

ttctatgtac tatttaaaaa acacaaactt ttggatgttc ggtttattct ttttctttta 360

cttttttatc atgggagcct acttcccgtt tttcccgatt tggctacatg acatcaacca 420

tatcagcaaa agtgatacgg gtattatttt tgccgctatt tctctgttct cgctattatt 480

ccaaccgctg tttggtctgc tttctgacaa actcgggctg cgcaaatacc tgctgtggat 540

tattaccggc atgttagtga tgtttgcgcc gttctttatt tttatcttcg ggccactgtt 600

acaatacaac attttagtag gatcgattgt tggtggtatt tatctaggct tttgttttaa 660

cgccggtgcg ccagcagtag aggcatttat tgagaaagtc agccgtcgca gtaatttcga 720

atttggtcgc gcgcggatgt ttggctgtgt tggctgggcg ctgtgtgcct cgattgtcgg 780

catcatgttc accatcaata atcagtttgt tttctggctg ggctctggct gtgcactcat 840

cctcgccgtt ttactctttt tcgccaaaac ggatgcgccc tcttctgcca cggttgccaa 900

tgcggtaggt gccaaccatt cggcatttag ccttaagctg gcactggaac tgttcagaca 960

gccaaaactg tggtttttgt cactgtatgt tattggcgtt tcctgcacct acgatgtttt 1020

tgaccaacag tttgctaatt tctttacttc gttctttgct accggtgaac agggtacgcg 1080

ggtatttggc tacgtaacga caatgggcga attacttaac gcctcgatta tgttctttgc 1140

gccactgatc attaatcgca tcggtgggaa aaacgccctg ctgctggctg gcactattat 1200

gtctgtacgt attattggct catcgttcgc cacctcagcg ctggaagtgg ttattctgaa 1260

aacgctgcat atgtttgaag taccgttcct gctggtgggc tgctttaaat atattaccag 1320

ccagtttgaa gtgcgttttt cagcgacgat ttatctggtc tgtttctgct tctttaagca 1380

actggcgatg atttttatgt ctgtactggc gggcaatatg tatgaaagca tcggtttcca 1440

gggcgcttat ctggtgctgg gtctggtggc gctgggcttc accttaattt ccgtgttcac 1500

gcttagcggc cccggcccgc tttccctgct gcgtcgtcag gtgaatgaag tcgcttaa 1558

<210> 17

<211> 1471

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 17

cccgtcttac tgtcgggaat tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat 60

tcgcgttggc cgattcttat taacaatatc cttcttgaca ttttgcaggg attgtgatat 120

aatcaataag tatgggccca agttcactta aaaaggagat caacaatgaa agcaattttc 180

gtactgaaac atcttaatca tgcgtaggat tttttctatg tactatttaa aaaacacaaa 240

cttttggatg ttcggtttat tctttttctt ttactttttt atcatgggag cctacttccc 300

gtttttcccg atttggctac atgacatcaa ccatatcagc aaaagtgata cgggtattat 360

ttttgccgct atttctctgt tctcgctatt attccaaccg ctgtttggtc tgctttctga 420

caaactcggg ctgcgcaaat acctgctgtg gattattacc ggcatgttag tgatgtttgc 480

gccgttcttt atttttatct tcgggccact gttacaatac aacattttag taggatcgat 540

tgttggtggt atttatctag gcttttgttt taacgccggt gcgccagcag tagaggcatt 600

tattgagaaa gtcagccgtc gcagtaattt cgaatttggt cgcgcgcgga tgtttggctg 660

tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg ttcaccatca ataatcagtt 720

tgttttctgg ctgggctctg gctgtgcact catcctcgcc gttttactct ttttcgccaa 780

aacggatgcg ccctcttctg ccacggttgc caatgcggta ggtgccaacc attcggcatt 840

tagccttaag ctggcactgg aactgttcag acagccaaaa ctgtggtttt tgtcactgta 900

tgttattggc gtttcctgca cctacgatgt ttttgaccaa cagtttgcta atttctttac 960

ttcgttcttt gctaccggtg aacagggtac gcgggtattt ggctacgtaa cgacaatggg 1020

cgaattactt aacgcctcga ttatgttctt tgcgccactg atcattaatc gcatcggtgg 1080

gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta cgtattattg gctcatcgtt 1140

cgccacctca gcgctggaag tggttattct gaaaacgctg catatgtttg aagtaccgtt 1200

cctgctggtg ggctgcttta aatatattac cagccagttt gaagtgcgtt tttcagcgac 1260

gatttatctg gtctgtttct gcttctttaa gcaactggcg atgattttta tgtctgtact 1320

ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct tatctggtgc tgggtctggt 1380

ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc ggccccggcc cgctttccct 1440

gctgcgtcgt caggtgaatg aagtcgctta a 1471

<210> 18

<211> 1632

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 18

cccgtcttac tgtcgggaat tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat 60

tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat tcgcgttggc caattaatta 120

atggtataag gttcttgaca tcttacaatc aatatggtat aataatttag ttagggccca 180

agttcactta aaaaggagat caacacttac tgtcgggaat tcgcgttggc caattaatta 240

atggtataag gttcttgaca tcttacaatc aatatggtat aaataattta gttagggccc 300

aagttcactt aaaaaggaga tcaacaatga aagcaatttt cgtactgaaa catcttaatc 360

atgcgtagga ttttttctat gtactattta aaaaacacaa acttttggat gttcggttta 420

ttctttttct tttacttttt tatcatggga gcctacttcc cgtttttccc gatttggcta 480

catgacatca accatatcag caaaagtgat acgggtatta tttttgccgc tatttctctg 540

ttctcgctat tattccaacc gctgtttggt ctgctttctg acaaactcgg gctgcgcaaa 600

tacctgctgt ggattattac cggcatgtta gtgatgtttg cgccgttctt tatttttatc 660

ttcgggccac tgttacaata caacatttta gtaggatcga ttgttggtgg tatttatcta 720

ggcttttgtt ttaacgccgg tgcgccagca gtagaggcat ttattgagaa agtcagccgt 780

cgcagtaatt tcgaatttgg tcgcgcgcgg atgtttggct gtgttggctg ggcgctgtgt 840

gcctcgattg tcggcatcat gttcaccatc aataatcagt ttgttttctg gctgggctct 900

ggctgtgcac tcatcctcgc cgttttactc tttttcgcca aaacggatgc gccctcttct 960

gccacggttg ccaatgcggt aggtgccaac cattcggcat ttagccttaa gctggcactg 1020

gaactgttca gacagccaaa actgtggttt ttgtcactgt atgttattgg cgtttcctgc 1080

acctacgatg tttttgacca acagtttgct aatttcttta cttcgttctt tgctaccggt 1140

gaacagggta cgcgggtatt tggctacgta acgacaatgg gcgaattact taacgcctcg 1200

attatgttct ttgcgccact gatcattaat cgcatcggtg ggaaaaacgc cctgctgctg 1260

gctggcacta ttatgtctgt acgtattatt ggctcatcgt tcgccacctc agcgctggaa 1320

gtggttattc tgaaaacgct gcatatgttt gaagtaccgt tcctgctggt gggctgcttt 1380

aaatatatta ccagccagtt tgaagtgcgt ttttcagcga cgatttatct ggtctgtttc 1440

tgcttcttta agcaactggc gatgattttt atgtctgtac tggcgggcaa tatgtatgaa 1500

agcatcggtt tccagggcgc ttatctggtg ctgggtctgg tggcgctggg cttcacctta 1560

atttccgtgt tcacgcttag cggccccggc ccgctttccc tgctgcgtcg tcaggtgaat 1620

gaagtcgctt aa 1632

<210> 19

<211> 1558

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 19

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcttactgt cgggaattcg cgttggccga ttcattaatg gtataaggtt 180

cttgacatct tacaatcaat atggtataat aatttagtta gggcccaagt tcacttaaaa 240

aggagatcaa caatgaaagc aattttcgta ctgaaacatc ttaatcatgc gtaggatttt 300

ttctatgtac tatttaaaaa acacaaactt ttggatgttc ggtttattct ttttctttta 360

cttttttatc atgggagcct acttcccgtt tttcccgatt tggctacatg acatcaacca 420

tatcagcaaa agtgatacgg gtattatttt tgccgctatt tctctgttct cgctattatt 480

ccaaccgctg tttggtctgc tttctgacaa actcgggctg cgcaaatacc tgctgtggat 540

tattaccggc atgttagtga tgtttgcgcc gttctttatt tttatcttcg ggccactgtt 600

acaatacaac attttagtag gatcgattgt tggtggtatt tatctaggct tttgttttaa 660

cgccggtgcg ccagcagtag aggcatttat tgagaaagtc agccgtcgca gtaatttcga 720

atttggtcgc gcgcggatgt ttggctgtgt tggctgggcg ctgtgtgcct cgattgtcgg 780

catcatgttc accatcaata atcagtttgt tttctggctg ggctctggct gtgcactcat 840

cctcgccgtt ttactctttt tcgccaaaac ggatgcgccc tcttctgcca cggttgccaa 900

tgcggtaggt gccaaccatt cggcatttag ccttaagctg gcactggaac tgttcagaca 960

gccaaaactg tggtttttgt cactgtatgt tattggcgtt tcctgcacct acgatgtttt 1020

tgaccaacag tttgctaatt tctttacttc gttctttgct accggtgaac agggtacgcg 1080

ggtatttggc tacgtaacga caatgggcga attacttaac gcctcgatta tgttctttgc 1140

gccactgatc attaatcgca tcggtgggaa aaacgccctg ctgctggctg gcactattat 1200

gtctgtacgt attattggct catcgttcgc cacctcagcg ctggaagtgg ttattctgaa 1260

aacgctgcat atgtttgaag taccgttcct gctggtgggc tgctttaaat atattaccag 1320

ccagtttgaa gtgcgttttt cagcgacgat ttatctggtc tgtttctgct tctttaagca 1380

actggcgatg atttttatgt ctgtactggc gggcaatatg tatgaaagca tcggtttcca 1440

gggcgcttat ctggtgctgg gtctggtggc gctgggcttc accttaattt ccgtgttcac 1500

gcttagcggc cccggcccgc tttccctgct gcgtcgtcag gtgaatgaag tcgcttaa 1558

<210> 20

<211> 1571

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 20

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaggctta cccgtcttac tgtcgggaat tcgcgttggc cgattcatta 180

atggtataag gttcttgaca tcttacaatc aatatggtat aataatttag ttagggccca 240

agttcactta aaaaggagat caacaatgaa agcaattttc gtactgaaac atcttaatca 300

tgcgtaggat tttttctatg tactatttaa aaaacacaaa cttttggatg ttcggtttat 360

tctttttctt ttactttttt atcatgggag cctacttccc gtttttcccg atttggctac 420

atgacatcaa ccatatcagc aaaagtgata cgggtattat ttttgccgct atttctctgt 480

tctcgctatt attccaaccg ctgtttggtc tgctttctga caaactcggg ctgcgcaaat 540

acctgctgtg gattattacc ggcatgttag tgatgtttgc gccgttcttt atttttatct 600

tcgggccact gttacaatac aacattttag taggatcgat tgttggtggt atttatctag 660

gcttttgttt taacgccggt gcgccagcag tagaggcatt tattgagaaa gtcagccgtc 720

gcagtaattt cgaatttggt cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg 780

cctcgattgt cggcatcatg ttcaccatca ataatcagtt tgttttctgg ctgggctctg 840

gctgtgcact catcctcgcc gttttactct ttttcgccaa aacggatgcg ccctcttctg 900

ccacggttgc caatgcggta ggtgccaacc attcggcatt tagccttaag ctggcactgg 960

aactgttcag acagccaaaa ctgtggtttt tgtcactgta tgttattggc gtttcctgca 1020

cctacgatgt ttttgaccaa cagtttgcta atttctttac ttcgttcttt gctaccggtg 1080

aacagggtac gcgggtattt ggctacgtaa cgacaatggg cgaattactt aacgcctcga 1140

ttatgttctt tgcgccactg atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg 1200

ctggcactat tatgtctgta cgtattattg gctcatcgtt cgccacctca gcgctggaag 1260

tggttattct gaaaacgctg catatgtttg aagtaccgtt cctgctggtg ggctgcttta 1320

aatatattac cagccagttt gaagtgcgtt tttcagcgac gatttatctg gtctgtttct 1380

gcttctttaa gcaactggcg atgattttta tgtctgtact ggcgggcaat atgtatgaaa 1440

gcatcggttt ccagggcgct tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa 1500

tttccgtgtt cacgcttagc ggccccggcc cgctttccct gctgcgtcgt caggtgaatg 1560

aagtcgctta a 1571

<210> 21

<211> 1431

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 21

cccgtcttac tgtcgggaat tcgcgttggc cgattcattt aaccgataag cttcttgaca 60

tgtttagggt gttatgatat aatcacccaa ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 180

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 240

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 300

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 360

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 420

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 480

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 540

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 600

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 660

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 720

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 780

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 840

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 900

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 960

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1020

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1080

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1140

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1200

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1260

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1320

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1380

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1431

<210> 22

<211> 1951

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 22

ggcacctgag tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag 60

tgaatggggg taaatggcac tacaggcgcc ttttatggat tcatgcaagg aaactaccca 120

taatacaaga aaagcccgtc acgggcttct cagggcgttt tatggcgggt ctgctatgtg 180

gtgctatctg actttttgct gttcagcagt tcctgccctc tgattttcca gtctgaccac 240

ttcggattat cccgtgacag gtcattcaga ctggctaatg cacccagtaa ggcagcggta 300

tcatcaacag gcttacccgt cttactgtcg ggaattcgcg ttggccgatt aaaggagatc 360

aacaatgaaa gcactgtcgg gaattcgcgt tggccgattc attaatggta taaggttctt 420

gacatcttac aatcaatatg gtataataat ttagttaggg cccaagttca cttaaaaagg 480

agatcaacaa tgaaagcaag cggtatcatc aacaggctta cccgtcttac tgtcgggaat 540

tcgcgttggc cgattcatta atggtataag gttcttgaca tcttacaatc aatatggtat 600

aataatttag ttagggccca agttcactta aaaaggagat caacaatgaa agcaattttc 660

gtactgaaac atcttaatca tgcgtaggat tttttctatg tactatttaa aaaacacaaa 720

cttttggatg ttcggtttat tctttttctt ttactttttt atcatgggag cctacttccc 780

gtttttcccg atttggctac atgacatcaa ccatatcagc aaaagtgata cgggtattat 840

ttttgccgct atttctctgt tctcgctatt attccaaccg ctgtttggtc tgctttctga 900

caaactcggg ctgcgcaaat acctgctgtg gattattacc ggcatgttag tgatgtttgc 960

gccgttcttt atttttatct tcgggccact gttacaatac aacattttag taggatcgat 1020

tgttggtggt atttatctag gcttttgttt taacgccggt gcgccagcag tagaggcatt 1080

tattgagaaa gtcagccgtc gcagtaattt cgaatttggt cgcgcgcgga tgtttggctg 1140

tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg ttcaccatca ataatcagtt 1200

tgttttctgg ctgggctctg gctgtgcact catcctcgcc gttttactct ttttcgccaa 1260

aacggatgcg ccctcttctg ccacggttgc caatgcggta ggtgccaacc attcggcatt 1320

tagccttaag ctggcactgg aactgttcag acagccaaaa ctgtggtttt tgtcactgta 1380

tgttattggc gtttcctgca cctacgatgt ttttgaccaa cagtttgcta atttctttac 1440

ttcgttcttt gctaccggtg aacagggtac gcgggtattt ggctacgtaa cgacaatggg 1500

cgaattactt aacgcctcga ttatgttctt tgcgccactg atcattaatc gcatcggtgg 1560

gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta cgtattattg gctcatcgtt 1620

cgccacctca gcgctggaag tggttattct gaaaacgctg catatgtttg aagtaccgtt 1680

cctgctggtg ggctgcttta aatatattac cagccagttt gaagtgcgtt tttcagcgac 1740

gatttatctg gtctgtttct gcttctttaa gcaactggcg atgattttta tgtctgtact 1800

ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct tatctggtgc tgggtctggt 1860

ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc ggccccggcc cgctttccct 1920

gctgcgtcgt caggtgaatg aagtcgctta a 1951

<210> 23

<211> 1749

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 23

cccgtcttac tgtcgggaat tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat 60

tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat 120

tcgcgttggc cgattcgaat tcgcgttggc cgattaaagg agatcaacaa tgaaagctta 180

ctgtcgggaa ttcgcgttgg ccgattcgaa ttcgcgttgg ccgattcatt aatggtataa 240

ggttcttgac atcttacaat caatatggta taataattta gttagggccc aagttcactt 300

aaaaaggaga tcaacaatga aagcttactg tcgggaattc gcgttggccg attcattaat 360

ggtataaggt tcttgacatc ttacaatcaa tatggtataa taatttagtt agggcccaag 420

ttcacttaaa aaggagatca acaatgaaag caattttcgt actgaaacat cttaatcatg 480

cgtaggattt tttctatgta ctatttaaaa aacacaaact tttggatgtt cggtttattc 540

tttttctttt acttttttat catgggagcc tacttcccgt ttttcccgat ttggctacat 600

gacatcaacc atatcagcaa aagtgatacg ggtattattt ttgccgctat ttctctgttc 660

tcgctattat tccaaccgct gtttggtctg ctttctgaca aactcgggct gcgcaaatac 720

ctgctgtgga ttattaccgg catgttagtg atgtttgcgc cgttctttat ttttatcttc 780

gggccactgt tacaatacaa cattttagta ggatcgattg ttggtggtat ttatctaggc 840

ttttgtttta acgccggtgc gccagcagta gaggcattta ttgagaaagt cagccgtcgc 900

agtaatttcg aatttggtcg cgcgcggatg tttggctgtg ttggctgggc gctgtgtgcc 960

tcgattgtcg gcatcatgtt caccatcaat aatcagtttg ttttctggct gggctctggc 1020

tgtgcactca tcctcgccgt tttactcttt ttcgccaaaa cggatgcgcc ctcttctgcc 1080

acggttgcca atgcggtagg tgccaaccat tcggcattta gccttaagct ggcactggaa 1140

ctgttcagac agccaaaact gtggtttttg tcactgtatg ttattggcgt ttcctgcacc 1200

tacgatgttt ttgaccaaca gtttgctaat ttctttactt cgttctttgc taccggtgaa 1260

cagggtacgc gggtatttgg ctacgtaacg acaatgggcg aattacttaa cgcctcgatt 1320

atgttctttg cgccactgat cattaatcgc atcggtggga aaaacgccct gctgctggct 1380

ggcactatta tgtctgtacg tattattggc tcatcgttcg ccacctcagc gctggaagtg 1440

gttattctga aaacgctgca tatgtttgaa gtaccgttcc tgctggtggg ctgctttaaa 1500

tatattacca gccagtttga agtgcgtttt tcagcgacga tttatctggt ctgtttctgc 1560

ttctttaagc aactggcgat gatttttatg tctgtactgg cgggcaatat gtatgaaagc 1620

atcggtttcc agggcgctta tctggtgctg ggtctggtgg cgctgggctt caccttaatt 1680

tccgtgttca cgcttagcgg ccccggcccg ctttccctgc tgcgtcgtca ggtgaatgaa 1740

gtcgcttaa 1749

<210> 24

<211> 1556

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 24

cccgtcttac tgtcgggaat tcgcgttggc cgattcatac taagcattat attcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcactgtcg ggaattcgcg ttggccaatt aattaatggt ataaggttct 180

tgacatctta caatcaatat ggtataataa tttagttagg gcccaagttc acttaaaaag 240

gagatcaaca atgaaagcaa ttttcgtact gaaacatctt aatcatgcgt aggatttttt 300

ctatgtacta tttaaaaaac acaaactttt ggatgttcgg tttattcttt ttcttttact 360

tttttatcat gggagcctac ttcccgtttt tcccgatttg gctacatgac atcaaccata 420

tcagcaaaag tgatacgggt attatttttg ccgctatttc tctgttctcg ctattattcc 480

aaccgctgtt tggtctgctt tctgacaaac tcgggctgcg caaatacctg ctgtggatta 540

ttaccggcat gttagtgatg tttgcgccgt tctttatttt tatcttcggg ccactgttac 600

aatacaacat tttagtagga tcgattgttg gtggtattta tctaggcttt tgttttaacg 660

ccggtgcgcc agcagtagag gcatttattg agaaagtcag ccgtcgcagt aatttcgaat 720

ttggtcgcgc gcggatgttt ggctgtgttg gctgggcgct gtgtgcctcg attgtcggca 780

tcatgttcac catcaataat cagtttgttt tctggctggg ctctggctgt gcactcatcc 840

tcgccgtttt actctttttc gccaaaacgg atgcgccctc ttctgccacg gttgccaatg 900

cggtaggtgc caaccattcg gcatttagcc ttaagctggc actggaactg ttcagacagc 960

caaaactgtg gtttttgtca ctgtatgtta ttggcgtttc ctgcacctac gatgtttttg 1020

accaacagtt tgctaatttc tttacttcgt tctttgctac cggtgaacag ggtacgcggg 1080

tatttggcta cgtaacgaca atgggcgaat tacttaacgc ctcgattatg ttctttgcgc 1140

cactgatcat taatcgcatc ggtgggaaaa acgccctgct gctggctggc actattatgt 1200

ctgtacgtat tattggctca tcgttcgcca cctcagcgct ggaagtggtt attctgaaaa 1260

cgctgcatat gtttgaagta ccgttcctgc tggtgggctg ctttaaatat attaccagcc 1320

agtttgaagt gcgtttttca gcgacgattt atctggtctg tttctgcttc tttaagcaac 1380

tggcgatgat ttttatgtct gtactggcgg gcaatatgta tgaaagcatc ggtttccagg 1440

gcgcttatct ggtgctgggt ctggtggcgc tgggcttcac cttaatttcc gtgttcacgc 1500

ttagcggccc cggcccgctt tccctgctgc gtcgtcaggt gaatgaagtc gcttaa 1556

<210> 25

<211> 1802

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 25

cccgtcttac tgtcgggaat tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat 60

tcgcgttggc cgattcgaat tcgcgttggc cgattaaagg agatcaacaa tgaaagctta 120

ctgtcgggaa ttcgcgttgg ccgattcatt aatggtataa ggttcttgac atcttacaat 180

caatatggta taataattta gttagggccc aagttcactt aaaaaggaga tcaacactta 240

ctgtcgggaa ttcgcgttgg ccaattaatt aatggtataa ggttcttgac atcttacaat 300

caatatggta taataattta gttagggccc aagttcactt aaaaaggaga tcaacaatga 360

aagcaggctt acccgtctta ctgtcgggaa ttcgcgttgg ccgattcatt aatggtataa 420

ggttcttgac atcttacaat caatatggta taataattta gttagggccc aagttcactt 480

aaaaaggaga tcaacaatga aagcaatttt cgtactgaaa catcttaatc atgcgtagga 540

ttttttctat gtactattta aaaaacacaa acttttggat gttcggttta ttctttttct 600

tttacttttt tatcatggga gcctacttcc cgtttttccc gatttggcta catgacatca 660

accatatcag caaaagtgat acgggtatta tttttgccgc tatttctctg ttctcgctat 720

tattccaacc gctgtttggt ctgctttctg acaaactcgg gctgcgcaaa tacctgctgt 780

ggattattac cggcatgtta gtgatgtttg cgccgttctt tatttttatc ttcgggccac 840

tgttacaata caacatttta gtaggatcga ttgttggtgg tatttatcta ggcttttgtt 900

ttaacgccgg tgcgccagca gtagaggcat ttattgagaa agtcagccgt cgcagtaatt 960

tcgaatttgg tcgcgcgcgg atgtttggct gtgttggctg ggcgctgtgt gcctcgattg 1020

tcggcatcat gttcaccatc aataatcagt ttgttttctg gctgggctct ggctgtgcac 1080

tcatcctcgc cgttttactc tttttcgcca aaacggatgc gccctcttct gccacggttg 1140

ccaatgcggt aggtgccaac cattcggcat ttagccttaa gctggcactg gaactgttca 1200

gacagccaaa actgtggttt ttgtcactgt atgttattgg cgtttcctgc acctacgatg 1260

tttttgacca acagtttgct aatttcttta cttcgttctt tgctaccggt gaacagggta 1320

cgcgggtatt tggctacgta acgacaatgg gcgaattact taacgcctcg attatgttct 1380

ttgcgccact gatcattaat cgcatcggtg ggaaaaacgc cctgctgctg gctggcacta 1440

ttatgtctgt acgtattatt ggctcatcgt tcgccacctc agcgctggaa gtggttattc 1500

tgaaaacgct gcatatgttt gaagtaccgt tcctgctggt gggctgcttt aaatatatta 1560

ccagccagtt tgaagtgcgt ttttcagcga cgatttatct ggtctgtttc tgcttcttta 1620

agcaactggc gatgattttt atgtctgtac tggcgggcaa tatgtatgaa agcatcggtt 1680

tccagggcgc ttatctggtg ctgggtctgg tggcgctggg cttcacctta atttccgtgt 1740

tcacgcttag cggccccggc ccgctttccc tgctgcgtcg tcaggtgaat gaagtcgctt 1800

aa 1802

<210> 26

<211> 1947

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 26

cctgagtcgc tgtctttttc gtgacattca gttcgctgcg ctcacggctc tggcagtgaa 60

tgggggtaaa tggcactaca ggcgcctttt atggattcat gcaaggaaac tacccataat 120

acaagaaaag cccgtcacgg gcttctcagg gcgttttatg gcgggtctgc tatgtggtgc 180

tatctgactt tttgctgttc agcagttcct gccctctgat tttccagtct gaccacttcg 240

gattatcccg tgacaggtca ttcagactgg ctaatgcacc cagtaaggca gcggtatcat 300

caacaggctt acccgtctta ctgtcgggaa ttcgcgttgg ccgattaaag gagatcaaca 360

atgaaagcac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 420

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 480

caacaatgaa agcaagcggt atcatcaaca ggcttacccg tcttactgtc gggaattcgc 540

gttggccgat tcattaatgg tataaggttc ttgacatctt acaatcaata tggcataata 600

atttagttag ggcccaagtt cacttaaaaa ggagatcaac aatgaaagca attttcgtac 660

tgaaacatct taatcatgcg taggattttt tctatgtact atttaaaaaa cacaaacttt 720

tggatgttcg gtttattctt tttcttttac ttttttatca tgggagccta cttcccgttt 780

ttcccgattt ggctacatga catcaaccat atcagcaaaa gtgatacggg tattattttt 840

gccgctattt ctctgttctc gctattattc caaccgctgt ttggtctgct ttctgacaaa 900

ctcgggctgc gcaaatacct gctgtggatt attaccggca tgttagtgat gtttgcgccg 960

ttctttattt ttatcttcgg gccactgtta caatacaaca ttttagtagg atcgattgtt 1020

ggtggtattt atctaggctt ttgttttaac gccggtgcgc cagcagtaga ggcatttatt 1080

gagaaagtca gccgtcgcag taatttcgaa tttggtcgcg cgcggatgtt tggctgtgtt 1140

ggctgggcgc tgtgtgcctc gattgtcggc atcatgttca ccatcaataa tcagtttgtt 1200

ttctggctgg gctctggctg tgcactcatc ctcgccgttt tactcttttt cgccaaaacg 1260

gatgcgccct cttctgccac ggttgccaat gcggtaggtg ccaaccattc ggcatttagc 1320

cttaagctgg cactggaact gttcagacag ccaaaactgt ggtttttgtc actgtatgtt 1380

attggcgttt cctgcaccta cgatgttttt gaccaacagt ttgctaattt ctttacttcg 1440

ttctttgcta ccggtgaaca gggtacgcgg gtatttggct acgtaacgac aatgggcgaa 1500

ttacttaacg cctcgattat gttctttgcg ccactgatca ttaatcgcat cggtgggaaa 1560

aacgccctgc tgctggctgg cactattatg tctgtacgta ttattggctc atcgttcgcc 1620

acctcagcgc tggaagtggt tattctgaaa acgctgcata tgtttgaagt accgttcctg 1680

ctggtgggct gctttaaata tattaccagc cagtttgaag tgcgtttttc agcgacgatt 1740

tatctggtct gtttctgctt ctttaagcaa ctggcgatga tttttatgtc tgtactggcg 1800

ggcaatatgt atgaaagcat cggtttccag ggcgcttatc tggtgctggg tctggtggcg 1860

ctgggcttca ccttaatttc cgtgttcacg cttagcggcc ccggcccgct ttccctgctg 1920

cgtcgtcagg tgaatgaagt cgcttaa 1947

<210> 27

<211> 1511

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 27

cccgtcttac tgtcgggaat tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat 60

tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat tcgcgttggc caattaatta 120

atggtataag gttcttgaca tcttacaatc aatatggtat aataatttag ttagggccca 180

agttcactta aaaaggagat caacaatgaa agcaattttc gtactgaaac atcttaatca 240

tgcgtaggat tttttctatg tactatttaa aaaacacaaa cttttggatg ttcggtttat 300

tctttttctt ttactttttt atcatgggag cctacttccc gtttttcccg atttggctac 360

atgacatcaa ccatatcagc aaaagtgata cgggtattat ttttgccgct atttctctgt 420

tctcgctatt attccaaccg ctgtttggtc tgctttctga caaactcggg ctgcgcaaat 480

acctgctgtg gattattacc ggcatgttag tgatgtttgc gccgttcttt atttttatct 540

tcgggccact gttacaatac aacattttag taggatcgat tgttggtggt atttatctag 600

gcttttgttt taacgccggt gcgccagcag tagaggcatt tattgagaaa gtcagccgtc 660

gcagtaattt cgaatttggt cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg 720

cctcgattgt cggcatcatg ttcaccatca ataatcagtt tgttttctgg ctgggctctg 780

gctgtgcact catcctcgcc gttttactct ttttcgccaa aacggatgcg ccctcttctg 840

ccacggttgc caatgcggta ggtgccaacc attcggcatt tagccttaag ctggcactgg 900

aactgttcag acagccaaaa ctgtggtttt tgtcactgta tgttattggc gtttcctgca 960

cctacgatgt ttttgaccaa cagtttgcta atttctttac ttcgttcttt gctaccggtg 1020

aacagggtac gcgggtattt ggctacgtaa cgacaatggg cgaattactt aacgcctcga 1080

ttatgttctt tgcgccactg atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg 1140

ctggcactat tatgtctgta cgtattattg gctcatcgtt cgccacctca gcgctggaag 1200

tggttattct gaaaacgctg catatgtttg aagtaccgtt cctgctggtg ggctgcttta 1260

aatatattac cagccagttt gaagtgcgtt tttcagcgac gatttatctg gtctgtttct 1320

gcttctttaa gcaactggcg atgattttta tgtctgtact ggcgggcaat atgtatgaaa 1380

gcatcggttt ccagggcgct tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa 1440

tttccgtgtt cacgcttagc ggccccggcc cgctttccct gctgcgtcgt caggtgaatg 1500

aagtcgctta a 1511

<210> 28

<211> 1950

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 28

cctgagtcgc tgtctttttc gtgacattca gttcgctgcg ctcacggctc tggcagtgaa 60

tgggggtaaa tggcactaca ggcgcctttt atggattcat gcaaggaaac tacccataat 120

acaagaaaag cccgtcacgg gcttctcagg gcgttttatg gcgggtctgc tatgtggtgc 180

tatctgactt tttgctgttc agcagttcct gccctctgat tttccagtct gaccacttcg 240

gattatcccg tgacaggtca ttcagactgg ctaatgcacc cagtaaggca gcggtatcat 300

caacaggctt acccgtctta ctgtcgggaa ttcgcgttgg ccgattaaag gagatcaaca 360

atgaaagcac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 420

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 480

caacaatgaa agcaagcggt atcatcaaca ggcttacccg tcttactgtc gggaattcgc 540

gttggccgat tcattaatgg tataaggttc tttgacatct tacaatcaat atggtataat 600

aatttagtta gggcccaagt tcacttaaaa aaggagatca acaatgaaag caattttcgt 660

actgaaacat cttaatcatg cgtaggattt ttttctatgt actatttaaa aaacacaaac 720

ttttggatgt tcggtttatt ctttttcttt tactttttta tcatgggagc ctacttcccg 780

tttttcccga tttggctaca tgacatcaac catatcagca aaagtgatac gggtattatt 840

tttgccgcta tttctctgtt ctcgctatta ttccaaccgc tgtttggtct gctttctgac 900

aaactcgggc tgcgcaaata cctgctgtgg attattaccg gcatgttagt gatgtttgcg 960

ccgttcttta tttttatctt cgggccactg ttacaataca acattttagt aggatcgatt 1020

gttggtggta tttatctagg cttttgtttt aacgccggtg cgccagcagt agaggcattt 1080

attgagaaag tcagccgtcg cagtaatttc gaatttggtc gcgcgcggat gtttggctgt 1140

gttggctggg cgctgtgtgc ctcgattgtc ggcatcatgt tcaccatcaa taatcagttt 1200

gttttctggc tgggctctgg ctgtgcactc atcctcgccg ttttactctt tttcgccaaa 1260

acggatgcgc cctcttctgc cacggttgcc aatgcggtag gtgccaacca ttcggcattt 1320

agccttaagc tggcactgga actgttcaga cagccaaaac tgtggttttt gtcactgtat 1380

gttattggcg tttcctgcac ctacgatgtt tttgaccaac agtttgctaa tttctttact 1440

tcgttctttg ctaccggtga acagggtacg cgggtatttg gctacgtaac gacaatgggc 1500

gaattactta acgcctcgat tatgttcttt gcgccactga tcattaatcg catcggtggg 1560

aaaaacgccc tgctgctggc tggcactatt atgtctgtac gtattattgg ctcatcgttc 1620

gccacctcag cgctggaagt ggttattctg aaaacgctgc atatgtttga agtaccgttc 1680

ctgctggtgg gctgctttaa atatattacc agccagtttg aagtgcgttt ttcagcgacg 1740

atttatctgg tctgtttctg cttctttaag caactggcga tgatttttat gtctgtactg 1800

gcgggcaata tgtatgaaag catcggtttc cagggcgctt atctggtgct gggtctggtg 1860

gcgctgggct tcaccttaat ttccgtgttc acgcttagcg gccccggccc gctttccctg 1920

ctgcgtcgtc aggtgaatga agtcgcttaa 1950

<210> 29

<211> 1551

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 29

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggcataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacacttac tgtcgggaat tcgcgttggc caattaatta atggtataag gttcttgaca 180

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 240

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtatgat tttttctatg 300

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 360

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 420

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 480

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 540

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 600

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 660

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 720

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 780

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 840

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 900

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 960

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 1020

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 1080

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1140

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1200

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1260

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1320

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1380

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1440

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1500

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1551

<210> 30

<211> 1431

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 30

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 180

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 240

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 300

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 360

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 420

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 480

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 540

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 600

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 660

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 720

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 780

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 840

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 900

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 960

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1020

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1080

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1140

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1200

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1260

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1320

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1380

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1431

<210> 31

<211> 1551

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 31

cccgtcttac tgtcgggaat tcgcgttggc cgattcattc aatccaatag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacacttac tgtcgggaat tcgcgttggc caattaatta atggtataag gttcttgaca 180

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 240

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 300

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 360

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 420

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 480

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 540

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 600

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 660

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 720

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 780

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 840

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 900

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 960

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 1020

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 1080

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1140

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1200

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1260

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1320

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1380

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1440

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1500

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1551

<210> 32

<211> 1558

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 32

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatgggat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcttactgt cgggaattcg cgttggccga ttcttataaa tcattatctt 180

cttgacattt tagaaacaat atggtataat ataacgataa gggcccaagt tcacttaaaa 240

aggagatcaa caatgaaagc aattttcgta ctgaaacatc ttaatcatgc gtaggatttt 300

ttctatgtac tatttaaaaa acacaaactt ttggatgttc ggtttattct ttttctttta 360

cttttttatc atgggagcct acttcccgtt tttcccgatt tggctacatg acatcaacca 420

tatcagcaaa agtgatacgg gtattatttt tgccgctatt tctctgttct cgctattatt 480

ccaaccgctg tttggtctgc tttctgacaa actcgggctg cgcaaatacc tgctgtggat 540

tattaccggc atgttagtga tgtttgcgcc gttctttatt tttatcttcg ggccactgtt 600

acaatacaac attttagtag gatcgattgt tggtggtatt tatctaggct tttgttttaa 660

cgccggtgcg ccagcagtag aggcatttat tgagaaagtc agccgtcgca gtaatttcga 720

atttggtcgc gcgcggatgt ttggctgtgt tggctgggcg ctgtgtgcct cgattgtcgg 780

catcatgttc accatcaata atcagtttgt tttctggctg ggctctggct gtgcactcat 840

cctcgccgtt ttactctttt tcgccaaaac ggatgcgccc tcttctgcca cggttgccaa 900

tgcggtaggt gccaaccatt cggcatttag ccttaagctg gcactggaac tgttcagaca 960

gccaaaactg tggtttttgt cactgtatgt tattggcgtt tcctgcacct acgatgtttt 1020

tgaccaacag tttgctaatt tctttacttc gttctttgct accggtgaac agggtacgcg 1080

ggtatttggc tacgtaacga caatgggcga attacttaac gcctcgatta tgttctttgc 1140

gccactgatc attaatcgca tcggtgggaa aaacgccctg ctgctggctg gcactattat 1200

gtctgtacgt attattggct catcgttcgc cacctcagcg ctggaagtgg ttattctgaa 1260

aacgctgcat atgtttgaag taccgttcct gctggtgggc tgctttaaat atattaccag 1320

ccagtttgaa gtgcgttttt cagcgacgat ttatctggtc tgtttctgct tctttaagca 1380

actggcgatg atttttatgt ctgtactggc gggcaatatg tatgaaagca tcggtttcca 1440

gggcgcttat ctggtgctgg gtctggtggc gctgggcttc accttaattt ccgtgttcac 1500

gcttagcggc cccggcccgc tttccctgct gcgtcgtcag gtgaatgaag tcgcttaa 1558

<210> 33

<211> 1542

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 33

cccgtcttac tgtcgggaat tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat 60

tcgcgctggc cgattcgaat tcgcgttggc cgattaaagg agatcaacta tgaaagctta 120

ctgtcgggaa ttcgcgttgg ccgattcatt aatggtataa ggctcttgac atcttacaat 180

caatatggta taataattta gttagggccc aagttcactt aaaaaggaga tcaacaatga 240

aagcaatttt cgtactgaaa catcttaatc atgcgtagga ttttttctat gtactattta 300

aaaaacacaa acttttggat gttcggttta ttctttttct tttacttttt tatcatggga 360

gcctacttcc cgtttttccc gatttggcta catgacatca accatatcag caaaagtgat 420

acgggtatta tttttgccgc tatttctctg ttctcgctat tattccaacc gctgtttggt 480

ctgctttctg acaaactcgg gctgcgcaaa tacctgctgt ggattattac cggcatgtta 540

gtgatgtttg cgccgttctt tatttttatc ttcgggccac tgttacaata caacatttta 600

gtaggatcga ttgttggtgg tatttatcta ggcttttgtt ttaacgccgg tgcgccagca 660

gtagaggcat ttattgagaa agtcagccgt cgcagtaatt tcgaatttgg tcgcgcgcgg 720

atgtttggct gtgttggctg ggcgctgtgt gcctcgattg tcggcatcat gttcaccatc 780

aataatcagt ttgttttctg gctgggctct ggctgtgcac tcatcctcgc cgttttactc 840

tttttcgcca aaacggatgc gccctcttct gccacggttg ccaatgcggt aggtgccaac 900

cattcggcat ttagccttaa gctggcactg gaactgttca gacagccaaa actgtggttt 960

ttgtcactgt atgttattgg cgtttcctgc acctacgatg tttttgacca acagtttgct 1020

aatttcttta cttcgttctt tgctaccggt gaacagggta cgcgggtatt tggctacgta 1080

acgacaatgg gcgaattact taacgcctcg attatgttct ttgcgccact gatcattaat 1140

cgcatcggtg ggaaaaacgc cctgctgctg gctggcacta ttatgtctgt acgtattatt 1200

ggctcatcgt tcgccacctc agcgctggaa gtggttattc tgaaaacgct gcatatgttt 1260

gaagtaccgt tcctgctggt gggctgcttt aaatatatta ccagccagtt tgaagtgcgt 1320

ttttcagcga cgatttatct ggtctgtttc tgcttcttta agcaactggc gatgattttt 1380

atgtctgtac tggcgggcaa tatgtatgaa agcatcggtt tccagggcgc ttatctggtg 1440

ctgggtctgg tggcgctggg cttcacctta atttccgtgt tcacgcttag cggccccggc 1500

ccgctttccc tgctgcgtcg tcaggtgaat gaagtcgctt aa 1542

<210> 34

<211> 1431

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 34

cccgtcttac tgtcgggaat tcgcgttggc cgattcatac tacccataat gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 180

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 240

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 300

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 360

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 420

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 480

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 540

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 600

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 660

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 720

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 780

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 840

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 900

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 960

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1020

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1080

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1140

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1200

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1260

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1320

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1380

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1431

<210> 35

<211> 1551

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 35

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacacttac tgtcgggaat tcgcgttggc caattaatta atggtataag gttcttgaca 180

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 240

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 300

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 360

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 420

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 480

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 540

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 600

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 660

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 720

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 780

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 840

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 900

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 960

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 1020

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 1080

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1140

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1200

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1260

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1320

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1380

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1440

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1500

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1551

<210> 36

<211> 1491

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 36

cccgtcttac tgtcgggaat tcgcgttggc cgattcgaat tcgcgatggc cgattcgaat 60

tcgcgttggc cgattcgaat tcgcgttggc cgattcttac acgatatata cttcttgaca 120

ttttgcggga attatggtat aatcacaagg ttagggccca agttcactta aaaaggagat 180

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 240

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 300

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 360

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 420

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 480

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 540

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 600

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 660

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 720

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 780

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 840

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 900

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 960

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 1020

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1080

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1140

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1200

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1260

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1320

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1380

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1440

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1491

<210> 37

<211> 1405

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 37

cccgtcttac tgtcgggaat tcgcgttggc caattaatta atggtataag gttcttgaca 60

tcttacaggg cccaagttca cttaaaaagg agatcaacaa tgaaagcaat tttcgtactg 120

aaacatctta atcatgcgta ggattttttc tatgtactat ttaaaaaaca caaacttttg 180

gatgttcggt ttattctttt tcttttactt ttttatcatg ggagcctact tcccgttttt 240

cccgatttgg ctacatgaca tcaaccatat cagcaaaagt gatacgggta ttatttttgc 300

cgctatttct ctgttctcgc tattattcca accgctgttt ggtctgcttt ctgacaaact 360

cgggctgcgc aaatacctgc tgtggattat taccggcatg ttagtgatgt ttgcgccgtt 420

ctttattttt atcttcgggc cactgttaca atacaacatt ttagtaggat cgattgttgg 480

tggtatttat ctaggctttt gttttaacgc cggtgcgcca gcagtagagg catttattga 540

gaaagtcagc cgtcgcagta atttcgaatt tggtcgcgcg cggatgtttg gctgtgttgg 600

ctgggcgctg tgtgcctcga ttgtcggcat catgttcacc atcaataatc agtttgtttt 660

ctggctgggc tctggctgtg cactcatcct cgccgtttta ctctttttcg ccaaaacgga 720

tgcgccctct tctgccacgg ttgccaatgc ggtaggtgcc aaccattcgg catttagcct 780

taagctggca ctggaactgt tcagacagcc aaaactgtgg tttttgtcac tgtatgttat 840

tggcgtttcc tgcacctacg atgtttttga ccaacagttt gctaatttct ttacttcgtt 900

ctttgctacc ggtgaacagg gtacgcgggt atttggctac gtaacgacaa tgggcgaatt 960

acttaacgcc tcgattatgt tctttgcgcc actgatcatt aatcgcatcg gtgggaaaaa 1020

cgccctgctg ctggctggca ctattatgtc tgtacgtatt attggctcat cgttcgccac 1080

ctcagcgctg gaagtggtta ttctgaaaac gctgcatatg tttgaagtac cgttcctgct 1140

ggtgggctgc tttaaatata ttaccagcca gtttgaagtg cgtttttcag cgacgattta 1200

tctggtctgt ttctgcttct ttaagcaact ggcgatgatt tttatgtctg tactggcggg 1260

caatatgtat gaaagcatcg gtttccaggg cgcttatctg gtgctgggtc tggtggcgct 1320

gggcttcacc ttaatttccg tgttcacgct tagcggcccc ggcccgcttt ccctgctgcg 1380

tcgtcaggtg aatgaagtcg cttaa 1405

<210> 38

<211> 1491

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 38

cccgtcttac tgtcgggaat tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat 60

tcgcgttggc cgattcgaat tcgcgttggc cgattcttac acgatatata cttcttgaca 120

ttttgcggga attatggtat aatcacaagg ttagggccca agttcactta aaaaggagat 180

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 240

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 300

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 360

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 420

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 480

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 540

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 600

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 660

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 720

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 780

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 840

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 900

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 960

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 1020

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1080

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1140

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1200

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1260

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1320

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1380

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1440

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1491

<210> 39

<211> 2116

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 39

cctgagtcgc tgtctttttc gtgacattca gttcgctgcg ctcacggctc tggcagtgaa 60

tgggggtaaa tggcactaca ggcgcctttt atggattcat gcaaggaaac tacccataat 120

acaagaaaag cccgtcacgg gcttctcagg gcgttttatg gcgggtctgc tatgtggtgc 180

tatctgactt tttgctgttc agcagttcct gccctctgat tttccagtct gaccacttcg 240

gattatcccg tgacaggtca ttcagactgg ctaatgcacc cagtaaggca gcggtatcat 300

caacaggctt acccgtctta ctgtcgggaa ttcgcgttgg ccgattcatt aatggtataa 360

ggttcttgac atcttacaat caatatggta taataattta gttagggccc aagttcactt 420

aaaaaggaga tcaacaatga aagcttactg tcgggaattc gcgttggccg attcattaat 480

ggtataaggt tcttgacatc ttacaatcaa tatggtataa taatttagtt agggcccaag 540

ttcacttaaa aaggagatca acaatgaaag cttactgtcg ggaattcgcg ttggccgatt 600

cattaatggt ataaggttct tgacatctta caatcaatat ggtataataa tttagttagg 660

gcccaagttc acttaaaaag gagatcaaca cttactgtcg ggaattcgcg ttggccaatt 720

aattaatggt ataaggttct tgacatctta caatcaatat ggtataataa tttagttagg 780

gcccaagttc acttaaaaag gagatcaaca atgaaagcaa ttttcgtact gaaacatctt 840

aatcatgcgt aggatttttt ctatgtacta tttaaaaaac acaaactttt ggatgttcgg 900

tttattcttt ttcttttact tttttatcat gggagcctac ttcccgtttt tcccgatttg 960

gctacatgac atcaaccata tcagcaaaag tgatacgggt attatttttg ccgctatttc 1020

tctgttctcg ctattattcc aaccgctgtt tggtctgctt tctgacaaac tcgggctgcg 1080

caaatacctg ctgtggatta ttaccggcat gttagtgatg tttgcgccgt tctttatttt 1140

tatcttcggg ccactgttac aatacaacat tttagtagga tcgattgttg gtggtattta 1200

tctaggcttt tgttttaacg ccggtgcgcc agcagtagag gcatttattg agaaagtcag 1260

ccgtcgcagt aatttcgaat ttggtcgcgc gcggatgttt ggctgtgttg gctgggcgct 1320

gtgtgcctcg attgtcggca tcatgttcac catcaataat cagtttgttt tctggctggg 1380

ctctggctgt gcactcatcc tcgccgtttt actctttttc gccaaaacgg atgcgccctc 1440

ttctgccacg gttgccaatg cggtaggtgc caaccattcg gcatttagcc ttaagctggc 1500

actggaactg ttcagacagc caaaactgtg gtttttgtca ctgtatgtta ttggcgtttc 1560

ctgcacctac gatgtttttg accaacagtt tgctaatttc tttacttcgt tctttgctac 1620

cggtgaacag ggtacgcggg tatttggcta cgtaacgaca atgggcgaat tacttaacgc 1680

ctcgattatg ttctttgcgc cactgatcat taatcgcatc ggtgggaaaa acgccctgct 1740

gctggctggc actattatgt ctgtacgtat tattggctca tcgttcgcca cctcagcgct 1800

ggaagtggtt attctgaaaa cgctgcatat gtttgaagta ccgttcctgc tggtgggctg 1860

ctttaaatat attaccagcc agtttgaagt gcgtttttca gcgacgattt atctggtctg 1920

tttctgcttc tttaagcaac tggcgatgat ttttatgtct gtactggcgg gcaatatgta 1980

tgaaagcatc ggtttccagg gcgcttatct ggtgctgggt ctggtggcgc tgggcttcac 2040

cttaatttcc gtgttcacgc ttagcggccc cggcccgctt tccctgctgc gtcgtcaggt 2100

gaatgaagtc gcttaa 2116

<210> 40

<211> 2122

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 40

tgtctttttc gtgacattca gttcgctgcg ctcacggctc tggcagtgaa tgggggtaaa 60

tggcactaca ggcgcctttt atggattcat gcaaggaaac tacccataat acaagaaaag 120

cccgtcacgg gcttctcagg gcgttttatg gcgggtctgc tatgtggtgc tatctgactt 180

tttgctgttc agcagttcct gccctctgat tttccagtct gaccacttcg gattatcccg 240

tgacaggtca ttcagactgg ctaatgcacc cagtaaggca gcggtatcat caacaggctt 300

acccgtctta ctgtcgggaa ttcgcgttgg ccgattcatt aatggtataa ggttcttgac 360

atcttacaat caatatggta taataattta gttagggccc aagttcactt aaaaaggaga 420

tcaacaatga aagcttactg tcgggaattc gcgttggccg attcattaat ggtataaggt 480

tcttgacatc ttacaatcaa tatggtataa taatttagtt agggcccaag ttcacttaaa 540

aaggagatca acaatgaaag cactgtcggg aattcgcgtt ggccgattca ttaatggtat 600

aaggttcttg acatcttaca atcaatatgg tataataatt tagttagggc ccaagttcac 660

ttaaaaagga gatcaacaat gaaagcttac tgtcgggaat tcgcgttggc cgattcatta 720

atggtataag gttcttgaca tcttacaatc aatatggtat aataatttag ttagggccca 780

agttcactta aaaaggagat caacacttta ctgtcgggga attcgccgtt gggccaattt 840

aattaatggg tataagggtt ccttgacaat gtactattta aaaaacacaa acttttggat 900

gttcggttta ttctttttct tttacttttt tatcatggga gcctacttcc cgtttttccc 960

gatttggcta catgacatca accatatcag caaaagtgat acgggtatta tttttgccgc 1020

tatttctctg ttctcgctat tattccaacc gctgtttggt ctgctttctg acaaactcgg 1080

gctgcgcaaa tacctgctgt ggattattac cggcatgtta gtgatgtttg cgccgttctt 1140

tatttttatc ttcgggccac tgttacaata caacatttta gtaggatcga ttgttggtgg 1200

tatttatcta ggcttttgtt ttaacgccgg tgcgccagca gtagaggcat ttattgagaa 1260

agtcagccgt cgcagtaatt tcgaatttgg tcgcgcgcgg atgtttggct gtgttggctg 1320

ggcgctgtgt gcctcgattg tcggcatcat gttcaccatc aataatcagt ttgttttctg 1380

gctgggctct ggctgtgcac tcatcctcgc cgttttactc tttttcgcca aaacggatgc 1440

gccctcttct gccacggttg ccaatgcggt aggtgccaac cattcggcat ttagccttaa 1500

gctggcactg gaactgttca gacagccaaa actgtggttt ttgtcactgt atgttattgg 1560

cgtttcctgc acctacgatg tttttgacca acagtttgct aatttcttta cttcgttctt 1620

tgctaccggt gaacagggta cgcgggtatt tggctacgta acgacaatgg gcgaattact 1680

taacgcctcg attatgttct ttgcgccact gatcattaat cgcatcggtg ggaaaaacgc 1740

cctgctgctg gctggcacta ttatgtctgt acgtattatt ggctcatcgt tcgccacctc 1800

agcgctggaa gtggttattc tgaaaacgct gcatatgttt gaagtaccgt tcctgctggt 1860

gggctgcttt aaatatatta ccagccagtt tgaagtgcgt ttttcagcga cgatttatct 1920

ggtctgtttc tgcttcttta agcaactggc gatgattttt atgtctgtac tggcgggcaa 1980

tatgtatgaa agcatcggtt tccagggcgc ttatctggtg ctgggtctgg tggcgctggg 2040

cttcacctta atttccgtgt tcacgcttag cggccccggc ccgctttccc tgctgcgtcg 2100

tcaggtgaat gaagtcgctt aa 2122

<210> 41

<211> 2130

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 41

tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag tgaatggggg 60

taaatggcac tacaggcgcc ttttatggat tcatgcaagg aaactaccca taatacaaga 120

aaagcccgtc acgggcttct cagggcgttt tatggcgggt ctgctatgtg gtgctatctg 180

actttttgct gttcagcagt tcctgccctc tgattttcca gtctgaccac ttcggattat 240

cccgtgacag gtcattcaga ctggctaatg cacccagtaa ggcagcggta tcatcaacag 300

gcttacccgt cttactgtcg ggaattcgcg ttggccgatt cattaatggt ataaggttct 360

tgacatctta caatcaatat ggtataataa tttagttagg gcccaagttc acttaaaaag 420

gagatcaaca atgaaagctt actgtcggga attcgcgttg gccgattcat taatggtata 480

aggttcttga catcttacaa tcaatatggt ataataattt agttagggcc caagttcact 540

taaaaaggag atcaacaatg aaagcactgt cgggaattcg cgttggccga ttcattaatg 600

gtataagggt cttgacatct tacaatcaat atggtataat aatttaggta ggggccaagt 660

tcacttagaa aggagatcaa caatgaaagc ttactgtcgg gaattcgcgt tggccgattc 720

attaatggta taaggttctt gacatcttac aatctatatg gtatcataat atacttaggg 780

cccaagttca cttaaaaagg agatcaacac ttactgtcgc gaattcgcgt tggccaatta 840

attcatggta taaggttctt gacatcttac aatcaaatgt actatttaaa aaacacaaac 900

ttttggatgt tcggtttatt ctttttcttt tactttttta tcatgggagc ctacttcccg 960

tttttcccga tttggctaca tgacatcaac catatcagca aaagtgatac gggtattatt 1020

tttgccgcta tttctctgtt ctcgctatta ttccaaccgc tgtttggtct gctttctgac 1080

aaactcgggc tgcgcaaata cctgctgtgg attattaccg gcatgttagt gatgtttgcg 1140

ccgttcttta tttttatctt cgggccactg ttacaataca acattttagt aggatcgatt 1200

gttggtggta tttatctagg cttttgtttt aacgccggtg cgccagcagt agaggcattt 1260

attgagaaag tcagccgtcg cagtaatttc gaatttggtc gcgcgcggat gtttggctgt 1320

gttggctggg cgctgtgtgc ctcgattgtc ggcatcatgt tcaccatcaa taatcagttt 1380

gttttctggc tgggctctgg ctgtgcactc atcctcgccg ttttactctt tttcgccaaa 1440

acggatgcgc cctcttctgc cacggttgcc aatgcggtag gtgccaacca ttcggcattt 1500

agccttaagc tggcactgga actgttcaga cagccaaaac tgtggttttt gtcactgtat 1560

gttattggcg tttcctgcac ctacgatgtt tttgaccaac agtttgctaa tttctttact 1620

tcgttctttg ctaccggtga acagggtacg cgggtatttg gctacgtaac gacaatgggc 1680

gaattactta acgcctcgat tatgttcttt gcgccactga tcattaatcg catcggtggg 1740

aaaaacgccc tgctgctggc tggcactatt atgtctgtac gtattattgg ctcatcgttc 1800

gccacctcag cgctggaagt ggttattctg aaaacgctgc atatgtttga agtaccgttc 1860

ctgctggtgg gctgctttaa atatattacc agccagtttg aagtgcgttt ttcagcgacg 1920

atttatctgg tctgtttctg cttctttaag caactggcga tgatttttat gtctgtactg 1980

gcgggcaata tgtatgaaag catcggtttc cagggcgctt atctggtgct gggtctggtg 2040

gcgctgggct tcaccttaat ttccgtgttc acgcttagcg gccccggccc gctttccctg 2100

ctgcgtcgtc aggtgaatga agtcgcttaa 2130

<210> 42

<211> 1865

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 42

gcccgtcacg ggcttctcag ggcgttttat ggcgggtctg ctatgtggtg ctatctgact 60

ttttgctgtt cagcagttcc tgccctctga ttttccagtc tgaccacttc ggattatccc 120

gtgacaggtc attcagactg gctaatgcac ccagtaaggc agcggtatca tcaacaggct 180

tacccgtctt actgtcggga attcgcgttg gccgattcat taatggtata aggttcttga 240

catcttacaa tcaatatggt ataataattt agttagggcc caagttcact taaaaaggag 300

atcaacaatg aaagcttact gtcgggaatt cgcgttggcc gattcattaa tggtataagg 360

ttcttgacat cttacaatca atatggtata ataatttagt tagggcccaa gttcacttaa 420

aaaggagatc aacaatgaaa gcactgtcgg gaattcgcgt tggccaatta attaatggta 480

taaggttctt gacatcttac aatcaatatg gtataataat ttagttaggg cccaagttca 540

cttaaaaagg agatcaacaa tgaaagcaat tttcgtactg aaacatctta atcatgcgta 600

ggattttttc tatgtactat ttaaaaaaca caaacttttg gatgttcggt ttattctttt 660

tcttttactt ttttatcatg ggagcctact tcccgttttt cccgatttgg ctacatgaca 720

tcaaccatat cagcaaaagt gatacgggta ttatttttgc cgctatttct ctgttctcgc 780

tattattcca accgctgttt ggtctgcttt ctgacaaact cgggctgcgc aaatacctgc 840

tgtggattat taccggcatg ttagtgatgt ttgcgccgtt ctttattttt atcttcgggc 900

cactgttaca atacaacatt ttagtaggat cgattgttgg tggtatttat ctaggctttt 960

gttttaacgc cggtgcgcca gcagtagagg catttattga gaaagtcagc cgtcgcagta 1020

atttcgaatt tggtcgcgcg cggatgtttg gctgtgttgg ctgggcgctg tgtgcctcga 1080

ttgtcggcat catgttcacc atcaataatc agtttgtttt ctggctgggc tctggctgtg 1140

cactcatcct cgccgtttta ctctttttcg ccaaaacgga tgcgccctct tctgccacgg 1200

ttgccaatgc ggtaggtgcc aaccattcgg catttagcct taagctggca ctggaactgt 1260

tcagacagcc aaaactgtgg tttttgtcac tgtatgttat tggcgtttcc tgcacctacg 1320

atgtttttga ccaacagttt gctaatttct ttacttcgtt ctttgctacc ggtgaacagg 1380

gtacgcgggt atttggctac gtaacgacaa tgggcgaatt acttaacgcc tcgattatgt 1440

tctttgcgcc actgatcatt aatcgcatcg gtgggaaaaa cgccctgctg ctggctggca 1500

ctattatgtc tgtacgtatt attggctcat cgttcgccac ctcagcgctg gaagtggtta 1560

ttctgaaaac gctgcatatg tttgaagtac cgttcctgct ggtgggctgc tttaaatata 1620

ttaccagcca gtttgaagtg cgtttttcag cgacgattta tctggtctgt ttctgcttct 1680

ttaagcaact ggcgatgatt tttatgtctg tactggcggg caatatgtat gaaagcatcg 1740

gtttccaggg cgcttatctg gtgctgggtc tggtggcgct gggcttcacc ttaatttccg 1800

tgttcacgct tagcggcccc ggcccgcttt ccctgctgcg tcgtcaggtg aatgaagtcg 1860

cttaa 1865

<210> 43

<211> 1802

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 43

gctgttcagc agttcctgcc ctctgatttt ccagtctgac cacttcggat tatcccgtga 60

caggtcattc agactggcta atgcacccag taaggcagcg gtatcatcaa caggcttacc 120

cgtcttactg tcgggaattc gcgttggccg attcattaat ggtataaggt tcttgacatc 180

ttacaatcaa tatggtataa taatttagtt agggcccaag ttcacttaaa aaggagatca 240

acaatgaaag cttactgtcg ggaattcgcg ttggccgatt cattaatggt ataaggttct 300

tgacatctta caatcaatat gggtataata atttagttag ggcccaagtt cacttaaaaa 360

ggagatcaac aatgaaagca ctgtcgggaa ttcgcgttgg ccgattcatt aatggtataa 420

ggttcttgac atcttacaat caatatggta taataattta gttagggccc aagttcactt 480

aaaaaggaga tcaacaatga aagcaatttt cgtactgaaa catcttaatc atgcgtagga 540

ttttttctat gtactattta aaaaacacaa acttttggat gttcggttta ttctttttct 600

tttacttttt tatcatggga gcctacttcc cgtttttccc gatttggcta catgacatca 660

accatatcag caaaagtgat acgggtatta tttttgccgc tatttctctg ttctcgctat 720

tattccaacc gctgtttggt ctgctttctg acaaactcgg gctgcgcaaa tacctgctgt 780

ggattattac cggcatgtta gtgatgtttg cgccgttctt tatttttatc ttcgggccac 840

tgttacaata caacatttta gtaggatcga ttgttggtgg tatttatcta ggcttttgtt 900

ttaacgccgg tgcgccagca gtagaggcat ttattgagaa agtcagccgt cgcagtaatt 960

tcgaatttgg tcgcgcgcgg atgtttggct gtgttggctg ggcgctgtgt gcctcgattg 1020

tcggcatcat gttcaccatc aataatcagt ttgttttctg gctgggctct ggctgtgcac 1080

tcatcctcgc cgttttactc tttttcgcca aaacggatgc gccctcttct gccacggttg 1140

ccaatgcggt aggtgccaac cattcggcat ttagccttaa gctggcactg gaactgttca 1200

gacagccaaa actgtggttt ttgtcactgt atgttattgg cgtttcctgc acctacgatg 1260

tttttgacca acagtttgct aatttcttta cttcgttctt tgctaccggt gaacagggta 1320

cgcgggtatt tggctacgta acgacaatgg gcgaattact taacgcctcg attatgttct 1380

ttgcgccact gatcattaat cgcatcggtg ggaaaaacgc cctgctgctg gctggcacta 1440

ttatgtctgt acgtattatt ggctcatcgt tcgccacctc agcgctggaa gtggttattc 1500

tgaaaacgct gcatatgttt gaagtaccgt tcctgctggt gggctgcttt aaatatatta 1560

ccagccagtt tgaagtgcgt ttttcagcga cgatttatct ggtctgtttc tgcttcttta 1620

agcaactggc gatgattttt atgtctgtac tggcgggcaa tatgtatgaa agcatcggtt 1680

tccagggcgc ttatctggtg ctgggtctgg tggcgctggg cttcacctta atttccgtgt 1740

tcacgcttag cggccccggc ccgctttccc tgctgcgtcg tcaggtgaat gaagtcgctt 1800

aa 1802

<210> 44

<211> 2245

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 44

ggcacctgag tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag 60

tgaatggggg taaatggcac tacaggcgcc ttttatggat tcatgcaagg aaactaccca 120

taatacaaga aaagcccgtc acgggcttct cagggcgttt tatggcgggt ctgctatgtg 180

gtgctatctg actttttgct gttcagcagt tcctgccctc tgattttcca gtctgaccac 240

ttcggattat cccgtgacag gtcattcaga ctggctaatg cacccagtaa ggcagcggta 300

tcatcaacag gcttacccgt cttactgtcg ggaattcgcg ttggccgatt cattaatggt 360

ataaggttct tgacatctta caatcaatat ggtataataa tttagttagg gcccaagttc 420

acttaaaaag gagatcaaca atgaaagctt actgtcggga attcgcgttg gccgattcat 480

taatggtata aggttcttga catcttacaa tcaatatggt ataataattt agttagggcc 540

caagttcact taaaaaggag atcaacaatg aaagcactgt cgggaattcg cgttggccga 600

ttcattaatg gtataaggtt cttgacatct tacaatcaat atggtataat aatttagtta 660

gggcccaagt tcacttaaaa aggagatcaa caatgaaagc ttactgtcgg gaattcgcgt 720

tggccgattc attaatggta taaggttctt gacatcttac aatcaatatg gtataataat 780

ttagttaggg cccaagttca cttaaaaagg agatcaacac ttactgtcgg gaattcgcgt 840

tggccaatta attaatggta taaggttctt gacatcttac aatcaatatg gtataataat 900

ttagttaggg cccaagttca cttaaaaagg agatcaacaa tgaaagcaat tttcgtactg 960

aaacatctta atcatgcgta ggattttttc tatgtactat ttaaaaaaca caaacttttg 1020

gatgttcggt ttattctttt tcttttactt ttttatcatg ggagcctact tcccgttttt 1080

cccgatttgg ctacatgaca tcaaccatat cagcaaaagt gatacgggta ttatttttgc 1140

cgctatttct ctgttctcgc tattattcca accgctgttt ggtctgcttt ctgacaaact 1200

cgggctgcgc aaatacctgc tgtggattat taccggcatg ttagtgatgt ttgcgccgtt 1260

ctttattttt atcttcgggc cactgttaca atacaacatt ttagtaggat cgattgttgg 1320

tggtatttat ctaggctttt gttttaacgc cggtgcgcca gcagtagagg catttattga 1380

gaaagtcagc cgtcgcagta atttcgaatt tggtcgcgcg cggatgtttg gctgtgttgg 1440

ctgggcgctg tgtgcctcga ttgtcggcat catgttcacc atcaataatc agtttgtttt 1500

ctggctgggc tctggctgtg cactcatcct cgccgtttta ctctttttcg ccaaaacgga 1560

tgcgccctct tctgccacgg ttgccaatgc ggtaggtgcc aaccattcgg catttagcct 1620

taagctggca ctggaactgt tcagacagcc aaaactgtgg tttttgtcac tgtatgttat 1680

tggcgtttcc tgcacctacg atgtttttga ccaacagttt gctaatttct ttacttcgtt 1740

ctttgctacc ggtgaacagg gtacgcgggt atttggctac gtaacgacaa tgggcgaatt 1800

acttaacgcc tcgattatgt tctttgcgcc actgatcatt aatcgcatcg gtgggaaaaa 1860

cgccctgctg ctggctggca ctattatgtc tgtacgtatt attggctcat cgttcgccac 1920

ctcagcgctg gaagtggtta ttctgaaaac gctgcatatg tttgaagtac cgttcctgct 1980

ggtgggctgc tttaaatata ttaccagcca gtttgaagtg cgtttttcag cgacgattta 2040

tctggtctgt ttctgcttct ttaagcaact ggcgatgatt tttatgtctg tactggcggg 2100

caatatgtat gaaagcatcg gtttccaggg cgcttatctg gtgctgggtc tggtggcgct 2160

gggcttcacc ttaatttccg tgttcacgct tagcggcccc ggcccgcttt ccctgctgcg 2220

tcgtcaggtg aatgaagtcg cttaa 2245

<210> 45

<211> 2029

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 45

gcaaggaaac tacccataat acaagaaaag cccgtcacgg gcttctcagg gcgttttatg 60

gcgggtctgc tatgtggtgc tatctgactt tttgctgttc agcagttcct gccctctgat 120

tttccagtct gaccacttcg gattatcccg tgacaggtca ttcagactgg ctaatgcacc 180

cagtaaggca gcggtatcat caacaggctt acccgtctta ctgtcgggaa ttcgcgttgg 240

ccgattaatt aatggtataa ggttcttgac atcttacaat caatatggta taataattta 300

gttagggccc aagttcactt aaaaaggaga tcaacaatga aagcttactg tcgggaattc 360

gcgttggccg attcattaat ggtataaggt tcttgacatc ttacaatcaa tatggtataa 420

taatttagtt agggcccaag ttcacttaaa aaggagatca acaatgaaag caggcttacc 480

cgtcttactg tcgggaattc gcgttggccg attcattaat ggtataaggt tcttgacatc 540

ttacaatcaa tatggtataa taatttagtt agggcccaag ttcacttaaa aaggagatca 600

acacttactg tcgggaattc gcgttggcca attaattaat ggtataaggt tcttgacatc 660

ttacaatcaa tatggtataa taatttagtt agggcccaag ttcacttaaa aaggagatca 720

acaatgaaag caattttcgt actgaaacat cttaatcatg cgtaggattt tttctatgta 780

ctatttaaaa aacacaaact tttggatgtt cggtttattc tttttctttt acttttttat 840

catgggagcc tacttcccgt ttttcccgat ttggctacat gacatcaacc atatcagcaa 900

aagtgatacg ggtattattt ttgccgctat ttctctgttc tcgctattat tccaaccgct 960

gtttggtctg ctttctgaca aactcgggct gcgcaaatac ctgctgtgga ttattaccgg 1020

catgttagtg atgtttgcgc cgttctttat ttttatcttc gggccactgt tacaatacaa 1080

cattttagta ggatcgattg ttggtggtat ttatctaggc ttttgtttta acgccggtgc 1140

gccagcagta gaggcattta ttgagaaagt cagccgtcgc agtaatttcg aatttggtcg 1200

cgcgcggatg tttggctgtg ttggctgggc gctgtgtgcc tcgattgtcg gcatcatgtt 1260

caccatcaat aatcagtttg ttttctggct gggctctggc tgtgcactca tcctcgccgt 1320

tttactcttt ttcgccaaaa cggatgcgcc ctcttctgcc acggttgcca atgcggtagg 1380

tgccaaccat tcggcattta gccttaagct ggcactggaa ctgttcagac agccaaaact 1440

gtggtttttg tcactgtatg ttattggcgt ttcctgcacc tacgatgttt ttgaccaaca 1500

gtttgctaat ttctttactt cgttctttgc taccggtgaa cagggtacgc gggtatttgg 1560

ctacgtaacg acaatgggcg aattacttaa cgcctcgatt atgttctttg cgccactgat 1620

cattaatcgc atcggtggga aaaacgccct gctgctggct ggcactatta tgtctgtacg 1680

tattattggc tcatcgttcg ccacctcagc gctggaagtg gttattctga aaacgctgca 1740

tatgtttgaa gtaccgttcc tgctggtggg ctgctttaaa tatattacca gccagtttga 1800

agtgcgtttt tcagcgacga tttatctggt ctgtttctgc ttctttaagc aactggcgat 1860

gatttttatg tctgtactgg cgggcaatat gtatgaaagc atcggtttcc agggcgctta 1920

tctggtgctg ggtctggtgg cgctgggctt caccttaatt tccgtgttca cgcttagcgg 1980

ccccggcccg ctttccctgc tgcgtcgtca ggtgaatgaa gtcgcttaa 2029

<210> 46

<211> 2088

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 46

ttcagttcgc tgcgctcacg gctctggcag tgaatggggg taaatggcac tacaggcgcc 60

ttttatggat tcatgcaagg aaactaccca taatacaaga aaagcccgtc acgggcttct 120

cagggcgttt tatggcgggt ctgctatgtg gtgctatctg actttttgct gttcagcagt 180

tcctgccctc tgattttcca gtctgaccac ttcggattat cccgtgacag gtcattcaga 240

ctggctaatg cacccagtaa ggcagcggta tcatcaacag gcttacccgt cttactgtcg 300

ggaattcgcg ttggccgatt cattaatggt ataaggttct tgacatctta caatcaatat 360

ggtataataa tttagttagg gcccaagttc acttaaaaag gagatcaaca atgaaagctt 420

actgtcggga attcgcgttg gccgattcat taatggtata aggttcttga catcttacaa 480

tcaatatggt ataataattt agttagggcc caagttcact taaaaaggag atcaacaatg 540

aaagcactgt cgggaattcg cgttggccga ttcattaatg gtataaggtt cttgacatct 600

tacaatcaat atggtataat aatttagtta gggcccaagt tcacttaaaa aggagatcaa 660

cacttactgt cgggaattcg cgttggccaa ttaattaatg gtataaggtt cttgacatct 720

tacaatcaat atggtataat aatttagtta gggcccaagt tcacttaaaa aggagatcaa 780

caatgaaagc aattttcgta ctgaaacatc ttaatcatgc gtaggatttt ttctatgtac 840

tatttaaaaa acacaaactt ttggatgttc ggtttattct ttttctttta cttttttatc 900

atgggagcct acttcccgtt tttcccgatt tggctacatg acatcaacca tatcagcaaa 960

agtgatacgg gtattatttt tgccgctatt tctctgttct cgctattatt ccaaccgctg 1020

tttggtctgc tttctgacaa actcgggctg cgcaaatacc tgctgtggat tattaccggc 1080

atgttagtga tgtttgcgcc gttctttatt tttatcttcg ggccactgtt acaatacaac 1140

attttagtag gatcgattgt tggtggtatt tatctaggct tttgttttaa cgccggtgcg 1200

ccagcagtag aggcatttat tgagaaagtc agccgtcgca gtaatttcga atttggtcgc 1260

gcgcggatgt ttggctgtgt tggctgggcg ctgtgtgcct cgattgtcgg catcatgttc 1320

accatcaata atcagtttgt tttctggctg ggctctggct gtgcactcat cctcgccgtt 1380

ttactctttt tcgccaaaac ggatgcgccc tcttctgcca cggttgccaa tgcggtaggt 1440

gccaaccatt cggcatttag ccttaagctg gcactggaac tgttcagaca gccaaaactg 1500

tggtttttgt cactgtatgt tattggcgtt tcctgcacct acgatgtttt tgaccaacag 1560

tttgctaatt tctttacttc gttctttgct accggtgaac agggtacgcg ggtatttggc 1620

tacgtaacga caatgggcga attacttaac gcctcgatta tgttctttgc gccactgatc 1680

attaatcgca tcggtgggaa aaacgccctg ctgctggctg gcactattat gtctgtacgt 1740

attattggct catcgttcgc cacctcagcg ctggaagtgg ttattctgaa aacgctgcat 1800

atgtttgaag taccgttcct gctggtgggc tgctttaaat atattaccag ccagtttgaa 1860

gtgcgttttt cagcgacgat ttatctggtc tgtttctgct tctttaagca actggcgatg 1920

atttttatgt ctgtactggc gggcaatatg tatgaaagca tcggtttcca gggcgcttat 1980

ctggtgctgg gtctggtggc gctgggcttc accttaattt ccgtgttcac gcttagcggc 2040

cccggcccgc tttccctgct gcgtcgtcag gtgaatgaag tcgcttaa 2088

<210> 47

<211> 2102

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 47

gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg 60

attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt 120

tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc 180

tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa 240

tgcacccagt aaggcagcgg tatcatcaac aggcttaccc gtcttactgt cgggaattcg 300

cgttggccga ttcgaattcg cgttggccga ttcgaattcg cgttggccga ttcgaattcg 360

cgttggccga ttcgaattcg cgttggccga ttcgaattcg cgttggccga ttcgaattcg 420

cgttggccga ttcgaattcg cgttggccga ttcattaatg gtataaggtt cttgacatct 480

tacaatcaat atggtataat aatttagtta gggcccaagt tcacttaaaa aggagatcaa 540

caatgaaagc ttactgtcgg gaattcgcgt tggccgattc attaatggta taaggttctt 600

gacatcttac aatcaatatg gtataataat ttagttaggg cccaagttca cttaaaaagg 660

agatcaacaa tgaaagctta ctgtcgggaa ttcgcgttgg ccgattcatt aatggtataa 720

ggttcttgac atcttacaat caatatggta taataattta gttagggccc aagttcactt 780

aaaaaggaga tcaacaatga aagcaatttt cgtactgaaa catcttaatc atgcgtagga 840

ttttttctat gtactattta aaaaacacaa acttttggat gttcggttta ttctttttct 900

tttacttttt tatcatggga gcctacttcc cgtttttccc gatttggcta catgacatca 960

accatatcag caaaagtgat acgggtatta tttttgccgc tatttctctg ttctcgctat 1020

tattccaacc gctgtttggt ctgctttctg acaaactcgg gctgcgcaaa tacctgctgt 1080

ggattattac cggcatgtta gtgatgtttg cgccgttctt tatttttatc ttcgggccac 1140

tgttacaata caacatttta gtaggatcga ttgttggtgg tatttatcta ggcttttgtt 1200

ttaacgccgg tgcgccagca gtagaggcat ttattgagaa agtcagccgt cgcagtaatt 1260

tcgaatttgg tcgcgcgcgg atgtttggct gtgttggctg ggcgctgtgt gcctcgattg 1320

tcggcatcat gttcaccatc aataatcagt ttgttttctg gctgggctct ggctgtgcac 1380

tcatcctcgc cgttttactc tttttcgcca aaacggatgc gccctcttct gccacggttg 1440

ccaatgcggt aggtgccaac cattcggcat ttagccttaa gctggcactg gaactgttca 1500

gacagccaaa actgtggttt ttgtcactgt atgttattgg cgtttcctgc acctacgatg 1560

tttttgacca acagtttgct aatttcttta cttcgttctt tgctaccggt gaacagggta 1620

cgcgggtatt tggctacgta acgacaatgg gcgaattact taacgcctcg attatgttct 1680

ttgcgccact gatcattaat cgcatcggtg ggaaaaacgc cctgctgctg gctggcacta 1740

ttatgtctgt acgtattatt ggctcatcgt tcgccacctc agcgctggaa gtggttattc 1800

tgaaaacgct gcatatgttt gaagtaccgt tcctgctggt gggctgcttt aaatatatta 1860

ccagccagtt tgaagtgcgt ttttcagcga cgatttatct ggtctgtttc tgcttcttta 1920

agcaactggc gatgattttt atgtctgtac tggcgggcaa tatgtatgaa agcatcggtt 1980

tccagggcgc ttatctggtg ctgggtctgg tggcgctggg cttcacctta atttccgtgt 2040

tcacgcttag cggccccggc ccgctttccc tgctgcgtcg tcaggtgaat gaagtcgctt 2100

aa 2102

<210> 48

<211> 2056

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<220>

<221> misc_feature

<222> (616) .. (616)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (656) .. (656)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (674) .. (674)

<223> n is a, c, g, or t

<400> 48

gctgttcagc agttcctgcc ctctgatttt ccagtctgac cacttcggat tatcccgtga 60

caggtcattc agactggcta atgcacccag taaggcagcg gtatcatcaa caggcttacc 120

cgtcttactg tcgggaattc gcgttggccg attaattaat ggtataaggt tcttgacatc 180

ttacaatcaa tatggtataa taatttagtt agggcccaag ttcacttaaa aaggagatca 240

acaatgaaag cttactgtcg ggaattcgcg ttggccgatt cattaatggt ataaggttct 300

tgacatctta caatcaatat ggtataataa tttagttagg gcccaagttc acttaaaaag 360

gagatcaaca atgaaagcag gcttacccgt cttactgtcg ggaattcgcg ttggccgatt 420

cattaatggt ataaggttct tgacatctta caatcaatat ggtataataa tttagttagg 480

gcccaagttc acttaaaaag gagatcaaca cttactgtcg ggaattcgcg ttggccaatt 540

aattaatggt ataaggttct tgacatctta caatcaatat ggtataataa tttagttagg 600

gcccaagttc acttanaaag gagatcaaca cttactgtcg ggaattcgcg ttggcncatt 660

aattaatggt atanagttct tgacatctta ccaatcatat ggtataataa tttagttagg 720

gcccaagttc acttaaaaag gagatcaaca atgaaagcaa ttttcgtact gaaacatctt 780

aatcatgcgt aggatttttt ctatgtacta tttaaaaaac acaaactttt ggatgttcgg 840

tttattcttt ttcttttact tttttatcat gggagcctac ttcccgtttt tcccgatttg 900

gctacatgac atcaaccata tcagcaaaag tgatacgggt attatttttg ccgctatttc 960

tctgttctcg ctattattcc aaccgctgtt tggtctgctt tctgacaaac tcgggctgcg 1020

caaatacctg ctgtggatta ttaccggcat gttagtgatg tttgcgccgt tctttatttt 1080

tatcttcggg ccactgttac aatacaacat tttagtagga tcgattgttg gtggtattta 1140

tctaggcttt tgttttaacg ccggtgcgcc agcagtagag gcatttattg agaaagtcag 1200

ccgtcgcagt aatttcgaat ttggtcgcgc gcggatgttt ggctgtgttg gctgggcgct 1260

gtgtgcctcg attgtcggca tcatgttcac catcaataat cagtttgttt tctggctggg 1320

ctctggctgt gcactcatcc tcgccgtttt actctttttc gccaaaacgg atgcgccctc 1380

ttctgccacg gttgccaatg cggtaggtgc caaccattcg gcatttagcc ttaagctggc 1440

actggaactg ttcagacagc caaaactgtg gtttttgtca ctgtatgtta ttggcgtttc 1500

ctgcacctac gatgtttttg accaacagtt tgctaatttc tttacttcgt tctttgctac 1560

cggtgaacag ggtacgcggg tatttggcta cgtaacgaca atgggcgaat tacttaacgc 1620

ctcgattatg ttctttgcgc cactgatcat taatcgcatc ggtgggaaaa acgccctgct 1680

gctggctggc actattatgt ctgtacgtat tattggctca tcgttcgcca cctcagcgct 1740

ggaagtggtt attctgaaaa cgctgcatat gtttgaagta ccgttcctgc tggtgggctg 1800

ctttaaatat attaccagcc agtttgaagt gcgtttttca gcgacgattt atctggtctg 1860

tttctgcttc tttaagcaac tggcgatgat ttttatgtct gtactggcgg gcaatatgta 1920

tgaaagcatc ggtttccagg gcgcttatct ggtgctgggt ctggtggcgc tgggcttcac 1980

cttaatttcc gtgttcacgc ttagcggccc cggcccgctt tccctgctgc gtcgtcaggt 2040

gaatgaagtc gcttaa 2056

<210> 49

<211> 1558

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 49

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcttactgt cgggaattcg cgttggccga ttcttataaa tcattatctt 180

cttgacattt tagaaacaat atggtataat ataacgataa gggcccaagt tcacttaaaa 240

aggagatcaa caatgaaagc aattttcgta ctgaaacatc ttaatcatgc gtaggatttt 300

ttctatgtac tatttaaaaa acacaaactt ttggatgttc ggtttattct ttttctttta 360

cttttttatc atgggagcct acttcccgtt tttcccgatt tggctacatg acatcaacca 420

tatcagcaaa agtgatacgg gtattatttt tgccgctatt tctctgttct cgctattatt 480

ccaaccgctg tttggtctgc tttctgacaa actcgggctg cgcaaatacc tgctgtggat 540

tattaccggc atgttagtga tgtttgcgcc gttctttatt tttatcttcg ggccactgtt 600

acaatacaac attttagtag gatcgattgt tggtggtatt tatctaggct tttgttttaa 660

cgccggtgcg ccagcagtag aggcatttat tgagaaagtc agccgtcgca gtaatttcga 720

atttggtcgc gcgcggatgt ttggctgtgt tggctgggcg ctgtgtgcct cgattgtcgg 780

catcatgttc accatcaata atcagtttgt tttctggctg ggctctggct gtgcactcat 840

cctcgccgtt ttactctttt tcgccaaaac ggatgcgccc tcttctgcca cggttgccaa 900

tgcggtaggt gccaaccatt cggcatttag ccttaagctg gcactggaac tgttcagaca 960

gccaaaactg tggtttttgt cactgtatgt tattggcgtt tcctgcacct acgatgtttt 1020

tgaccaacag tttgctaatt tctttacttc gttctttgct accggtgaac agggtacgcg 1080

ggtatttggc tacgtaacga caatgggcga attacttaac gcctcgatta tgttctttgc 1140

gccactgatc attaatcgca tcggtgggaa aaacgccctg ctgctggctg gcactattat 1200

gtctgtacgt attattggct catcgttcgc cacctcagcg ctggaagtgg ttattctgaa 1260

aacgctgcat atgtttgaag taccgttcct gctggtgggc tgctttaaat atattaccag 1320

ccagtttgaa gtgcgttttt cagcgacgat ttatctggtc tgtttctgct tctttaagca 1380

actggcgatg atttttatgt ctgtactggc gggcaatatg tatgaaagca tcggtttcca 1440

gggcgcttat ctggtgctgg gtctggtggc gctgggcttc accttaattt ccgtgttcac 1500

gcttagcggc cccggcccgc tttccctgct gcgtcgtcag gtgaatgaag tcgcttaa 1558

<210> 50

<211> 1431

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 50

cccgtcttac tgtcgggaat tcgcgttggc cgattcattt aaccgataag cttcttgaca 60

tgtttagggt gttatgatat aatcacccaa ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 180

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 240

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 300

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 360

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 420

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 480

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 540

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 600

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 660

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 720

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 780

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 840

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 900

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 960

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1020

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1080

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1140

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1200

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1260

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1320

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1380

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1431

<210> 51

<211> 1431

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 51

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 180

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 240

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 300

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 360

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 420

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 480

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 540

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 600

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 660

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 720

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 780

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 840

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 900

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 960

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1020

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1080

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1140

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1200

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1260

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1320

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1380

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1431

<210> 52

<211> 1431

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 52

cccgtcttac tgtcgggaat tcgcgttggc cgattcttaa attcaataac tttcttgaca 60

tgttattaga tttatggtat aatgacccga tatgggccca agttcactta aaaaggagat 120

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 180

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 240

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 300

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 360

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 420

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 480

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 540

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 600

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 660

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 720

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 780

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 840

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 900

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 960

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1020

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1080

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1140

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1200

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1260

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1320

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1380

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1431

<210> 53

<211> 1571

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 53

cccgtcttac tgtcgggaat tcgcgttggc cgattaatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaggctta cccgtcttac tgtcgggaat tcgcgttggc caattaatta 180

atggtataag gttcttgaca tcttacaatc aatatggtat aataatttag ttagggccca 240

agttcactta aaaaggagat caacaatgaa agcaattttc gtactgaaac atcttaatca 300

tgcgtaggat tttttctatg tactatttaa aaaacacaaa cttttggatg ttcggtttat 360

tctttttctt ttactttttt atcatgggag cctacttccc gtttttcccg atttggctac 420

atgacatcaa ccatatcagc aaaagtgata cgggtattat ttttgccgct atttctctgt 480

tctcgctatt attccaaccg ctgtttggtc tgctttctga caaactcggg ctgcgcaaat 540

acctgctgtg gattattacc ggcatgttag tgatgtttgc gccgttcttt atttttatct 600

tcgggccact gttacaatac aacattttag taggatcgat tgttggtggt atttatctag 660

gcttttgttt taacgccggt gcgccagcag tagaggcatt tattgagaaa gtcagccgtc 720

gcagtaattt cgaatttggt cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg 780

cctcgattgt cggcatcatg ttcaccatca ataatcagtt tgttttctgg ctgggctctg 840

gctgtgcact catcctcgcc gttttactct ttttcgccaa aacggatgcg ccctcttctg 900

ccacggttgc caatgcggta ggtgccaacc attcggcatt tagccttaag ctggcactgg 960

aactgttcag acagccaaaa ctgtggtttt tgtcactgta tgttattggc gtttcctgca 1020

cctacgatgt ttttgaccaa cagtttgcta atttctttac ttcgttcttt gctaccggtg 1080

aacagggtac gcgggtattt ggctacgtaa cgacaatggg cgaattactt aacgcctcga 1140

ttatgttctt tgcgccactg atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg 1200

ctggcactat tatgtctgta cgtattattg gctcatcgtt cgccacctca gcgctggaag 1260

tggttattct gaaaacgctg catatgtttg aagtaccgtt cctgctggtg ggctgcttta 1320

aatatattac cagccagttt gaagtgcgtt tttcagcgac gatttatctg gtctgtttct 1380

gcttctttaa gcaactggcg atgattttta tgtctgtact ggcgggcaat atgtatgaaa 1440

gcatcggttt ccagggcgct tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa 1500

tttccgtgtt cacgcttagc ggccccggcc cgctttccct gctgcgtcgt caggtgaatg 1560

aagtcgctta a 1571

<210> 54

<211> 1431

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 54

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 180

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 240

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 300

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 360

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 420

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 480

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 540

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 600

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 660

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 720

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 780

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 840

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 900

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 960

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1020

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1080

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1140

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1200

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1260

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1320

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1380

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1431

<210> 55

<211> 1968

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 55

cccgtcttac tgtcgggaat tcgcgttggc cgattcgaat tcgcgttggc cgattcgaat 60

tcgcgttggc cgattcgaat tcgcgttggc cgattcatta atggtataag gttcttgaca 120

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 180

caacacttac tgtcgggaat tcgcgttggc cgattcatta atggtataag gttcttgaca 240

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 300

caacaatgaa agcttactgt cgggaattcg cgttggccga ttcattaatg gtataaggtt 360

cttgacatct tacaatcaat atggtataat aatttagtta gggcccaagt tcacttaaaa 420

aggagatcaa caatgaaagc ttactgtcgg gaattcgcgt tggccgattc attaatggta 480

taaggttctt gacatcttac aatcaatatg gtataataat ttagttaggg cccaagttca 540

cttaaaaagg agatcaacac ttactgtcgg gaattcgcgt tggccaatta attaatggta 600

taaggttctt gacatcttac catcaatatg gggcccaagt tcacttaaaa aggagatcaa 660

caatgaaagc aattttcgta ctgaaacatc ttaatcatgc gtaggatttt ttctatgtac 720

tatttaaaaa acacaaactt ttggatgttc ggtttattct ttttctttta cttttttatc 780

atgggagcct acttcccgtt tttcccgatt tggctacatg acatcaacca tatcagcaaa 840

agtgatacgg gtattatttt tgccgctatt tctctgttct cgctattatt ccaaccgctg 900

tttggtctgc tttctgacaa actcgggctg cgcaaatacc tgctgtggat tattaccggc 960

atgttagtga tgtttgcgcc gttctttatt tttatcttcg ggccactgtt acaatacaac 1020

attttagtag gatcgattgt tggtggtatt tatctaggct tttgttttaa cgccggtgcg 1080

ccagcagtag aggcatttat tgagaaagtc agccgtcgca gtaatttcga atttggtcgc 1140

gcgcggatgt ttggctgtgt tggctgggcg ctgtgtgcct cgattgtcgg catcatgttc 1200

accatcaata atcagtttgt tttctggctg ggctctggct gtgcactcat cctcgccgtt 1260

ttactctttt tcgccaaaac ggatgcgccc tcttctgcca cggttgccaa tgcggtaggt 1320

gccaaccatt cggcatttag ccttaagctg gcactggaac tgttcagaca gccaaaactg 1380

tggtttttgt cactgtatgt tattggcgtt tcctgcacct acgatgtttt tgaccaacag 1440

tttgctaatt tctttacttc gttctttgct accggtgaac agggtacgcg ggtatttggc 1500

tacgtaacga caatgggcga attacttaac gcctcgatta tgttctttgc gccactgatc 1560

attaatcgca tcggtgggaa aaacgccctg ctgctggctg gcactattat gtctgtacgt 1620

attattggct catcgttcgc cacctcagcg ctggaagtgg ttattctgaa aacgctgcat 1680

atgtttgaag taccgttcct gctggtgggc tgctttaaat atattaccag ccagtttgaa 1740

gtgcgttttt cagcgacgat ttatctggtc tgtttctgct tctttaagca actggcgatg 1800

atttttatgt ctgtactggc gggcaatatg tatgaaagca tcggtttcca gggcgcttat 1860

ctggtgctgg gtctggtggc gctgggcttc accttaattt ccgtgttcac gcttagcggc 1920

cccggcccgc tttccctgct gcgtcgtcag gtgaatgaag tcgcttaa 1968

<210> 56

<211> 1431

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 56

cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atgggataag gttcttgaca 60

tcttacaatc aatatggtat aataatttag ttagggccca agttcactta aaaaggagat 120

caacaatgaa agcaattttc gtactgaaac atcttaatca tgcgtaggat tttttctatg 180

tactatttaa aaaacacaaa cttttggatg ttcggtttat tctttttctt ttactttttt 240

atcatgggag cctacttccc gtttttcccg atttggctac atgacatcaa ccatatcagc 300

aaaagtgata cgggtattat ttttgccgct atttctctgt tctcgctatt attccaaccg 360

ctgtttggtc tgctttctga caaactcggg ctgcgcaaat acctgctgtg gattattacc 420

ggcatgttag tgatgtttgc gccgttcttt atttttatct tcgggccact gttacaatac 480

aacattttag taggatcgat tgttggtggt atttatctag gcttttgttt taacgccggt 540

gcgccagcag tagaggcatt tattgagaaa gtcagccgtc gcagtaattt cgaatttggt 600

cgcgcgcgga tgtttggctg tgttggctgg gcgctgtgtg cctcgattgt cggcatcatg 660

ttcaccatca ataatcagtt tgttttctgg ctgggctctg gctgtgcact catcctcgcc 720

gttttactct ttttcgccaa aacggatgcg ccctcttctg ccacggttgc caatgcggta 780

ggtgccaacc attcggcatt tagccttaag ctggcactgg aactgttcag acagccaaaa 840

ctgtggtttt tgtcactgta tgttattggc gtttcctgca cctacgatgt ttttgaccaa 900

cagtttgcta atttctttac ttcgttcttt gctaccggtg aacagggtac gcgggtattt 960

ggctacgtaa cgacaatggg cgaattactt aacgcctcga ttatgttctt tgcgccactg 1020

atcattaatc gcatcggtgg gaaaaacgcc ctgctgctgg ctggcactat tatgtctgta 1080

cgtattattg gctcatcgtt cgccacctca gcgctggaag tggttattct gaaaacgctg 1140

catatgtttg aagtaccgtt cctgctggtg ggctgcttta aatatattac cagccagttt 1200

gaagtgcgtt tttcagcgac gatttatctg gtctgtttct gcttctttaa gcaactggcg 1260

atgattttta tgtctgtact ggcgggcaat atgtatgaaa gcatcggttt ccagggcgct 1320

tatctggtgc tgggtctggt ggcgctgggc ttcaccttaa tttccgtgtt cacgcttagc 1380

ggccccggcc cgctttccct gctgcgtcgt caggtgaatg aagtcgctta a 1431

<210> 57

<211> 2296

<212> DNA

<213> Artificial sequence

<220>

<223> Heterologous sequences in front of a lactose transporter gene

<400> 57

ggcacctgag tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag 60

tgaatggggg taaatggcac tacaggcgcc ttttatggat tcatgcaagg aaactaccca 120

taatacaaga aaagcccgtc acgggcttct cagggcgttt tatggcgggt ctgctatgtg 180

gtgctatctg actttttgct gttcagcagt tcctgccctc tgattttcca gtctgaccac 240

ttcggattat cccgtgacag gtcattcaga ctggctaatg cacccagtaa ggcagcggta 300

tcatcaacag gcttacccgt cttactgtcg ggaattcgcg ttggccgatt cgaattcgcg 360

ttggccgatt cttagagata aaaaatttct tgacatgttc tatgttttgt gatataatcg 420

tgagataagg gcccaagttc acttaaaaag gagatcaaca atgaaagcaa gcggtatcat 480

caacaggctt acccgtctta ctgtcgggaa ttcgcgttgg ccgattcatt aatggtataa 540

ggttcttgac atcttacaat caatatggta taataattta gttagggccc aagttcactt 600

aaaaaggaga tcaacaatga aagcttactg tcgggaattc gcgttggccg attcattaat 660

ggtataaggt tcttgacatc ttacaatcaa tatggtataa taatttagtt agggcccaag 720

ttcacttaaa aaggagatca acaatgaaag cttactgtcg ggaattcgcg ttggccgatt 780

cattaatggt ataaggttct tgacatctta caatcaatat ggtataataa tttagttagg 840

gcccaagttc acttaaaaag gagatcaaca cttactgtcg ggaattcgcg ttggccaatt 900

aattaatggt ataaggttct tgacatctta caatcaatat tgtataataa tttagttagg 960

gcccaagttc acttaaaaag gagatcaaca atgaaagcaa ttttcgtact gaaacatctt 1020

aatcatgcgt aggatttttt ctatgtacta tttaaaaaac acaaactttt ggatgttcgg 1080

tttattcttt ttcttttact tttttatcat gggagcctac ttcccgtttt tcccgatttg 1140

gctacatgac atcaaccata tcagcaaaag tgatacgggt attatttttg ccgctatttc 1200

tctgttctcg ctattattcc aaccgctgtt tggtctgctt tctgacaaac tcgggctgcg 1260

caaatacctg ctgtggatta ttaccggcat gttagtgatg tttgcgccgt tctttatttt 1320

tatcttcggg ccactgttac aatacaacat tttagtagga tcgattgttg gtggtattta 1380

tctaggcttt tgttttaacg ccggtgcgcc agcagtagag gcatttattg agaaagtcag 1440

ccgtcgcagt aatttcgaat ttggtcgcgc gcggatgttt ggctgtgttg gctgggcgct 1500

gtgtgcctcg attgtcggca tcatgttcac catcaataat cagtttgttt tctggctggg 1560

ctctggctgt gcactcatcc tcgccgtttt actctttttc gccaaaacgg atgcgccctc 1620

ttctgccacg gttgccaatg cggtaggtgc caaccattcg gcatttagcc ttaagctggc 1680

actggaactg ttcagacagc caaaactgtg gtttttgtca ctgtatgtta ttggcgtttc 1740

ctgcacctac gatgtttttg accaacagtt tgctaatttc tttacttcgt tctttgctac 1800

cggtgaacag ggtacgcggg tatttggcta cgtaacgaca atgggcgaat tacttaacgc 1860

ctcgattatg ttctttgcgc cactgatcat taatcgcatc ggtgggaaaa acgccctgct 1920

gctggctggc actattatgt ctgtacgtat tattggctca tcgttcgcca cctcagcgct 1980

ggaagtggtt attctgaaaa cgctgcatat gtttgaagta ccgttcctgc tggtgggctg 2040

ctttaaatat attaccagcc agtttgaagt gcgtttttca gcgacgattt atctggtctg 2100

tttctgcttc tttaagcaac tggcgatgat ttttatgtct gtactggcgg gcaatatgta 2160

tgaaagcatc ggtttccagg gcgcttatct ggtgctgggt ctggtggcgc tgggcttcac 2220

cttaatttcc gtgttcacgc ttagcggccc cggcccgctt tccctgctgc gtcgtcaggt 2280

gaatgaagtc gcttaa 2296

<210> 58

<211> 2210

<212> DNA

<213> Artificial sequence

<220>

<223> Terminator construct

<400> 58

tcgcgttggc cgattcggaa taggacacca tcgaatggtg caaaaccttt cgcggtatgg 60

catgatagcg cccggaagag agtcaattca gggtggtgaa tatgtactat ttaaaaaaca 120

caaacttttg gatgttcggt ttattctttt tcttttactt ttttatcatg ggagcctact 180

tcccgttttt cccgatttgg ctacatgaca tcaaccatat cagcaaaagt gatacgggta 240

ttatttttgc cgctatttct ctgttctcgc tattattcca accgctgttt ggtctgcttt 300

ctgacaaact cgggctgcgc aaatacctgc tgtggattat taccggcatg ttagtgatgt 360

ttgcgccgtt ctttattttt atcttcgggc cactgttaca atacaacatt ttagtaggat 420

cgattgttgg tggtatttat ctaggctttt gttttaacgc cggtgcgcca gcagtagagg 480

catttattga gaaagtcagc cgtcgcagta atttcgaatt tggtcgcgcg cggatgtttg 540

gctgtgttgg ctgggcgctg tgtgcctcga ttgtcggcat catgttcacc atcaataatc 600

agtttgtttt ctggctgggc tctggctgtg cactcatcct cgccgtttta ctctttttcg 660

ccaaaacgga tgcgccctct tctgccacgg ttgccaatgc ggtaggtgcc aaccattcgg 720

catttagcct taagctggca ctggaactgt tcagacagcc aaaactgtgg tttttgtcac 780

tgtatgttat tggcgtttcc tgcacctacg atgtttttga ccaacagttt gctaatttct 840

ttacttcgtt ctttgctacc ggtgaacagg gtacgcgggt atttggctac gtaacgacaa 900

tgggcgaatt acttaacgcc tcgattatgt tctttgcgcc actgatcatt aatcgcatcg 960

gtgggaaaaa cgccctgctg ctggctggca ctattatgtc tgtacgtatt attggctcat 1020

cgttcgccac ctcagcgctg gaagtggtta ttctgaaaac gctgcatatg tttgaagtac 1080

cgttcctgct ggtgggctgc tttaaatata ttaccagcca gtttgaagtg cgtttttcag 1140

cgacgattta tctggtctgt ttctgcttct ttaagcaact ggcgatgatt tttatgtctg 1200

tactggcggg caatatgtat gaaagcatcg gtttccaggg cgcttatctg gtgctgggtc 1260

tggtggcgct gggcttcacc ttaatttccg tgttcacgct tagcggcccc ggcccgcttt 1320

ccctgctgcg tcgtcaggtg aatgaagtcg ctcatcatca ccaccatcat taggatggtg 1380

gtgatgataa tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat 1440

cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt 1500

cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg 1560

gcctttattc acattcttgc ccgcctgatg aatgctcatc cggagttccg tatggcaatg 1620

aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag 1680

caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta 1740

cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg 1800

tttattgaga atatgttttt cgtcagcgcc aatccctggg tgagtttcac cagttttgat 1860

ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcactatggg caaatattat 1920

acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat 1980

ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc 2040

ggggcgtaag gatccaaagg tacctctaga gtcgacctgc aggccttcgt aaatctggcg 2100

agtggggaac tgccagacat caaataaaac aaaaggctca gtcggaagac tgggcctttt 2160

gttttatctg ttgtttgtcg gtgaacactc tcccgtgtag gctggagctg 2210

<---

Claims (24)

1. A method of obtaining microorganisms resistant to the phenomenon of suppression by the action of lactose, grown in an environment in which lactose is combined with another carbon source, where the method includes: a. altering the expression of the lactose transporter in microorganisms by introducing a heterologous promoter before the endogenous or exogenous gene of the lactose transporter or by changing the codons of the endogenous lactose transporter gene to codons used by the microorganism more often, or to codons used by the microorganism less often, b. growing said microorganisms on a medium containing a carbon source other than lactose, c. adding lactose to said medium, including a carbon source other than lactose, during the growth of said microorganisms, and d. detecting the expression of the lactose transporter, and e. selection of microorganisms resistant to the phenomenon of suppression by the action of lactose, growing on the specified medium, including lactose and another carbon source, which retain at least 50% of the level of lactose transport into the cell, achieved with the wild-type expression cassette of the specified lactose transporter, based on detected expression of the lactose transporter, wherein said carbon source other than lactose is selected from the group consisting of glycerol, maltose, glucose, fructose, sucrose, fucose, mannose, sialic acid, starch, cellulose, polyols, organic acids and pentoses. 2. The method according to claim 1, where the introduction of a heterologous promoter in front of the endogenous or exogenous gene of the lactose transporter is carried out by a) deletion of endogenous lactose transporters from the genome and introducing them into another region in the genome of said microorganism, or b) by introducing a heterologous promoter before endogenous lactose transporters, or c) by knocking out an endogenous lactose promoter and introducing a heterologous promoter into the same region of the genome of said microorganism. 3. The method according to PP. 1, 2, wherein said expressed lactose transporter is detected by translational binding to a reporter gene and / or by binding to an aptamer. 4. The method according to PP. 1-3, wherein said expressed lactose transporter is detected by the genetic constructs set forth in SEQ ID NOs 2, 3, 4 and / or 5. 5. The method according to PP. 1, 2, wherein said expressed lactose transporter is detected by measuring the growth rate of mutated microorganisms. 6. The method according to PP. 1, 2, wherein said expressed lactose transporter is detected by measuring the growth rate of mutated microorganisms on a medium containing lactose as the sole carbon source. 7. The method according to PP. 1-6, wherein said lactose transporter is lactose permease. 8. The method according to PP. 1-7, where the specified microorganism is a bacterial, yeast or fungal cell. 9. Microorganism resistant to the phenomenon of lactose inhibition, grown in an environment in which lactose is combined with other carbon source (s) selected from the group consisting of glycerin, maltose, glucose, fructose, sucrose, fucose , mannose, sialic acid, starch, cellulose, polyols, organic acids and pentoses, and the specified microorganism is obtained by the method according to PP. 1-8, and wherein said microorganism contains a heterologous promoter in front of an endogenous or exogenous lactose transporter gene or an endogenous lactose transporter gene, in which codons are changed to codons used by the microorganism more often, or to codons used by the microorganism less often. 10. Microorganism resistant to the phenomenon of lactose suppression, grown in an environment in which lactose is combined with other carbon source (s) selected from the group consisting of glycerin, maltose, glucose, fructose, sucrose, fucose , mannose, sialic acid, starch, cellulose, polyols, organic acids and pentoses containing a lactose transporter expression cassette containing the sequence shown in SEQ ID NOs 1, 7, 8, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57, and the specified microorganism is obtained by the method according to PP. 1-8. 11. Microorganism according to PP. 9, 10, in which genes encoding enzymes from lactose and / or galactose degradation pathways are less functional than a functional wild-type gene or non-functional. 12. A lactose transporter expression cassette resulting in the expression of a lactose transporter and which does not lead to a lactose inhibition phenotype, said lactose transporter expression cassette being obtained by isolation from a microorganism according to claim 10, and wherein said expression cassette contains the sequence shown in SEQ ID NO 1, 7, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or 57. 13. The use of a microorganism according to any one of claims. 9-11 or an expression cassette of a lactose transporter according to claim 12 for the production of 2-fucosyllactose, 2'-fucosyllactose, 3-fucosyllactose, 2 ', 3-difucosyllactose, lacto-N-triose, lacto-N-tetraose, lacto-nN- tetraose, 3'-sialyllactose, 6'-sialyllactose, glycolipids or galactosylated compounds. 14. A method of obtaining an expression cassette of a lactose transporter, which does not lead to the phenotype of suppression by the action of lactose, including: - creation of mutants of the lactose transporter expression cassette by introducing a heterologous promoter in front of the lactose transporter gene or by changing the codons of the lactose transporter gene to codons used by the microorganism more often, or to codons used by the microorganism less often, and these mutants demonstrate altered expression of the lactose transporter gene as compared to the corresponding wild-type lactose transporter gene; - screening mutant expression cassettes for the presence of expression cassettes that provide the expression of the lactose transporter in a microorganism transformed with said expression cassette; - screening mutant expression cassettes that provide expression of the lactose transporter for the presence of expression cassettes that do not lead to suppression by the action of lactose in a microorganism transformed by said expression cassette by growing microorganisms transformed with said expression cassettes in a medium including a carbon source other than lactose, and addition of lactose during the middle of the exponential phase, wherein said carbon source other than lactose is selected from the group consisting of glycerol, maltose, glucose, fructose, sucrose, fucose, mannose, sialic acid, starch, cellulose, polyols, organic acids and pentoses ; and - isolating said expression cassette from said transformed microorganisms which (i) are resistant to the phenomenon of lactose inhibition and which (ii) retain at least 50% of the level of lactose transport into the cell achieved with the wild-type expression cassette of said lactose transporter.

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